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Protein Topology, Sorting and Phosphorylation in Mitochondria

by

Nancy Anne Elizabeth Steenaart

A thesis submitted to the Faculty of Graduate Stutics and Research in partial fulfillment of the requirements of the degree of Doctor of Philosophy.

Department of Biochernisuy

McGill University, Montreal

Quebec, Canada

O Nancy A.E. Steenaart, October, 1997.

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ABSTRACT

The specificity of protein import into mitochondria is dependent on several

interacting factors. These include the phospholipids and the receptors of both membranes

as well as the targeting signals found within the precursor proteins themselves. The

targeting signals within precursor proteins were investigated in order to elucidate the role of

positively charged amino acids and the length and arnphiphilicity of the hydrophilic region

of a signal-anchor in conferring the orientation of that signal-anchor in the outer membrane

of the mitochondrion. It was determined that the retention of the hydrophilic region of the

signal-anchor on the cytosolic side of the outer membrane is due to the amphiphilicity of

this region and not due to the net positive charge or length of this region. The amphiphilic

region has lipid binding capabilities which are decreased upon reducing the hydrophobic

moment of this region.

The transmembrane region of a signal-anchor was further investigated to determine

how stretches of hydrophobic amino acids within precursor proteins function to target

preproteins to different locations within the mitochondrion. It was determined that the

targeting of a hydrophobic region is dependent on several factors: 1) its net hydrophobicity,

2) whether or not it is permitted to cross the outer membrane, 3) its distance from a matrix-

targeting signal and 4) the relative strength of the matrix-targeting signal. Specifically, the

mean hydrophobicity of the potential transmembrane region is an important determinant for

the insertion of bitopic proteins into the inner membrane of mitochondria.

Novel rnitochondrial proteins which are potential targets for phosphorylation were

also investigated. Cytochrome c oxidase subunit IV was found to be phosphorylated by an

endogenous kinase which is present in both whole mitochondria and in isolated

rnitoc hondrial membranes.

La spCcificitC de "l'importation protCiqueU (ciblage et insertion) ii l'interieur des

mitochondries relbve de plusieurs facteurs interdependants. Ces facteurs comprennent ies

phospholipides ainsi que les ricepteun des deux membranes mitochondriales, de meme

que les signaux de ciblage retrouvis dans les prodines pr6curseurs elles memes (sdquences

d'ancrage). Nous avons CtudiC les signaux de ciblage j. meme les proteines pricurseurs.

afin d'klucider le rdle des acides-aminis charges positivement, ansi que la longueur et le

caractbre "amphiphilique" de la region hydrophile d'un signal d'ancrage, dam la

determination de I'orientation de cette siquence d'ancrage l'intdrieur de la membrane

externe de la mitochondrie. Les rksultats obtenus dkmontrent que la retention de la region

hydrophile de la sequence d'ancrage sur la face cytosolique de la membrane externe lors de

I'insertion protCique, est dtterminie par le caractere "amphiphilique" de la region et non par

la charge positive nette ou la longueur de cette rdgion. De plus, la rkgion "amphiphilique"

posskde la capacitk de lier les lipides membranaires, capacite diminuant avec la riduction du

caract5re "arnphiphilique" de cette rCgion.

Une itude plus approfondie de la dgion trans-membranaire d'une sequence

d'ancrage a permis de cerner le mCcanisme d'action des acides-aminks hydrophobes j.

meme les protiines precurseurs, dans le ciblage des prCprotCines aux diffkrents

emplacements h I'indrieur de la mitochondrie. Les r6sultats obtenus dkmontrent que le

ciblage de la rigion hydrophobe est dkpendant de plusieurs facteurs: 1) l'hydrophobiciti

nette de cette region, 2) du fait que cette region puissc ou non traverser la membrane

exteme, 3) la distance de cettc region par rapport ii une sequence de ciblage i la matrice, et

4) la "force" relative de la sequence de ciblage 5 la matrice. Plus spicifiquement,

lrhydrophobicitC moyenne de la r6gion trans-membranaire potentielle constitue un

determinant important pour l'insertion de protkines renfermant un seul domaine trans-

rnembranaire, h I'inte'rieur de la membrane interne des mitochondries.

De nouvelles protkines mitochondriales, constituants des cibles potentielles de

phosphorylation, ont it6 tgalement investiguees. Ces Ctudes ont dbmontrees que la sous-

unid IV du cytochrome c oxydase est phosphorylte par une protkine kinase endogtne,

presente dam les mitochondries entitres, ainsi que dam des fractions membranaires.

To John for oil of his love and encoirmgernent

and to

Christopher Sean

nnJ

Lianne Kaitlin

for the excitement and lalcghter they create.

ACKNOWLEDGMENTS

I especially wish to thank my supervisor, Dr. Gordon C. Shore, for his guidance,

encouragement, kindness and support throughout my degree.

I also would like to thank all of the members of the Shore laboratory throughout the

years who have provided a very enjoyable and stimulating place to work. These include

Radka Bouchkova, Charles Boulakia, Lee Boyer, Gang Chen, Naila Chughtai, Ing Swie

Goping, Monique LagaccC, Sonia Lamontagne, Maribeth Lazarro. Jimmy Li. Josie Lavoie.

Irene Lee Rivera, Heidi McBride, Doug Millar, Munhy Madiraju, Flo Ng, Mai Nguyen.

Rathna Raju, Enrico Schleiff, and Mary Sutherland. I especially wish to thank my "bay-

mate" for d l of these years, Ing Swie Goping, for being a good friend and my resource for

molecular biology; Rathna Raju for looking after my well-being; Mai Nguyen for keeping

the lab lively; Sonia Lamontagne, Ing Swie Goping, Heidi McBride, and Flo Ng for

outings to Thomson house and downtown.

I also wish to thank the other professors of the McGill Department of Biochemistry

who have shown interest in my work and provided me with help and encouragement, and

Maureen Caron and Marlene Gilhooly for ail of their help.

Special thanks to Dr. JosCe Lavoie for translating my abstract into French.

I wish to thank the Fonds pour la Formation de Chercheur et I'Aide h la Recherche

(FCAR) and the McGill Department of Medicine for support in the form of studentships.

I wish to thank all of my friends for their interest and support, and of course I want

to thank all of the Steenaarts and Orlowskis for their love and encouragement.

vii

PREFACE

In accordance with the regulations stated in the Guidelines for thesis preparation of

the Faculty of Graduate Studies and Research of McGill University, the following

paragraphs are reproduced in full:

"Candidates have the option of including, as part of the thesis, the text of one or more

papers submitted or to be submitted for publication, or the clearly-duplicated text of one or

more published papers. These texts must be bound as an integral part of the thesis.

If this option is chosen, connecting texts that provide logical bridges between the different papers are mandatory. The thesis must be written in such a way that it

is more than a collection of manuscripts; in other words, results of a series of papers must

be integrated.

The thesis must still conform to all other requirements of the "Guidelines for Thesis

Preparation". The thesis must include: A Table of Contents, an abstract in English

and French, an introduction which clearly states the rationale and objectives of the study. a

comprehensive review of the literature, a final conclusion and summary, and a thorough

bibliography or reference list.

Additional material must be provided where appropriate (e.g. in appendices) and in

sufficient detail to allow a clear and precise judgment to be made of the importance and

originality of the research reported in the thesis.

In the case of manuscripts co-authored by the candidate and others, the candidate is required to make an explicit statement in the thesis as to who contributed to such work and to what extent. Supervisors must attest to the accuracy of such

statements at the doctoral oral defense. Since the task of the examiners is made more

difficult in these cases, it is in the candidate's interest to make perfectly clear the

responsibilities of all the authors of the co-authored papers. Under no circumstances can a co-author of any component of such a thesis serve as an examiner for that thesis."

As approved by the Department of Biochemistry, three manuscripts which have

been published have been included in this thesis.

Chapter 2: Steenaart, N.A.E., Silvius, J.R. and Shore, G.C. (1996) An Amphiphilic Lipid-Binding Domain Influences the Topology of a Signal-Anchor

Sequence in the Mitochondrial Outer Membrane. Biochemistry 35,3764-

377 1.

Chapter 3: Steenaart, N.A.E. and Shore, G.C. (1997) Alteration of a Mitochondrial

Signal Anchor Sequence That Permits Its Insertion into the Inner

Membrane. Contribution of Hydrophobic Residues. J. Bid . Chem. 272, 12057- 1206 1,

Chapter 4: Steenam, N.A.E. and Shore, G.C. (1997) Mitochondria1 Cytochrome c

Oxidase Subunit IV is Phosphorylated by an Endogenous Kinase. FEBS Letts. 415, 294-298.

In Chapter 2, Dr. John Silvius provided me with the liposomes which were used in

Figure 6; and in Chapter 4, Figure 1 was performed by P.H. Cameron. Other than these

exceptions. all of the work described in these three manuscripts is entirely my own.

TABLE OF CONTENTS

ABSTRACT RESUME

DEDICATION

ACKNOWLEDGMENTS

PREFACE

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER 1: htroduc tion

1.1 Mitochondria

1.2 Mitochondria1 Impon

1 -23 Overview

1.2b Cy tosolic Chaperones

1.22 Translocase of the Outer Mitochondrid Membrane (Tom)

1.2d Translocase of the Inner Mitochondrial Membrane (Tim)

1.2e Matrix Chaperones

1.2f Energetics

1 2 - 1 ATP Requirement 1.2-2 A y Requirement

1.2g Processing Proteases

1.3 Topogenic Import Signals

1.3a Matrix-Targeting Signals

1.3b Outer Membrane Signals

11 . . . LLl

v

vi

vii

ix xii xiv

1 . 3 ~ Inner Membrane and Intermembrane Space Signals 2 1

1.3d Protein Sorting between Membranes 23 1.4 Phosphorylation and Mitochondria 24

1.4a Kinases and Phosphoproteins in Mitochondria

1.5 Rationale and Objectives

CHAPTER 2: An Amphiphilic Lipid-Binding Domain Influences the Topology 29

of a Signal-Anchor Sequence in the Mitochondria1 Outer Membrane.

Abstract

lntroduc tion

Materials and Methods

Results and Discussion

Conclusions

Acknowledgments

References

CHAPTER 3: Alteration of a Mitochondria1 Outer Membrane Signal Anchor 30

Sequence That Permits Its Insertion into the Inner Membrane.

Contribution of Hydrophobic Residues.

Abstract

In~oduction

Materids and Methods

Results and Discussion

Conclusions

Acknowledgments

References

CHAPTER 4:

Mitoc hondrial Cytochrome c Oxidase Subunit IV is Phosphorylated 3 1

by an Endogenous Kinase.

Abstract

Introduction

Materials and Methods Results and Discussion

Conclusions

Acknowledgments

References

CHAPTER 5: General Discussion

5.1 Summary

5.2 Orientation of Signal-Anchor Sequences

5.3 Inner Membrane Targeting

5.4 Systems to Determine Tom and Tim Function

5.5 Phosphorylation of COX IV 5.6 Future Directions

L 1

REFERENCES

ORIGINAL CONTRIBUTIONS TO KNOWLEDGE

APPENDIX: Construction of Plasmids used in Chapters 2 and 3.

A. I Constructs-Chapter 2

A.2 Constructs-Chapter 3

LIST OF FIGURES

CHAPTER 1:

Figrire 1 : Protein import into mitochondria.

CHAPTER 2:

Figure I : pOMD29 and PO-OMD. 29-3766

Figure 2: Schematic illustration of structural alterations to the NH2- 29-3766

terminal hydrophilic domain of the signal-anchor sequence

of pOMD29 and PO-OMD.

Figwe 3: Insertion of pOMD29 and PO-OMD into the mitochondria1 29-3767

outer membrane.

Figure 4: import of pOMD29 and pO-OMD constructs lacking a 29-3768

transmembrane segment.

Figure 5: PO-OMD KR4QS. 29-3768

Figrirr 6: Liposome binding of PO-OMD KR4 A and PO-OMD 29-3769

KRJQS A.

Figrrre 7: Orientation of signal-anchor sequences in the

mitochondrial outer membrane.

CHAPTER 3:

Figure I : Fusion protein constructs. 30- 12058

Figure 2: Import of pO-SA 141, PO- 14 1 A, and pO-DHm into 30- 12059

intact mitochondria.

Figure 3: Import of PO-SA 14 1, p0- 14 1 A, PO-DKFR, PO-SA 36, 30- 12059

and pOMD29 into mitoplasts.

Figlire 4: Mutations in the yTom70p domain of PO-SA 14 1 that 30- 12060

permit insertion into the mitochondria1 inner membrane.

Figure 5: Schematic of the location and orientation of the various 30- 12060

fusion proteins following import into mitochondria (A)

and mitoplasts (B).

CHAPTER 4:

Figure 1: Pattern of ~ 2 ~ - ~ h o s ~ h o r ~ l a t e d proteins in microsomes, 38a

mitochondria and mitochondria1 membranes.

Figure 2: Purification of pp 17: DEAE-Sepharose chromatography. 38b

Figlire 3: Purification of pp 17: Preparatory gel electrophoresis. 39a

Figure I: Comparison of trypsin generated peptide sequence with 39b

cytochrome c oxidase subunit IV.

Figure 5: Identification of pp 17 as cytochrome c oxidase subunit 40a

IV.

xiv

LIST OF ABBREVIATIONS

2D

A'v ail

ADP ATP

bp CAMP

CCCP

COOH

COX

COX ny Da

DHFR DNA

ER Hsp70

IM r.MP IMS kDsi

Mdj 1

mGrpE

mHsp 10

mHsp60 rnHsp70

MIP MPP

MSF

mtDNA M T S NCBR

N. crassa NH2

two-dimensional membrane potential

amino acid

adenosine diphosphate

adenosine triphosphate

base pairs

3',5'-cyclic adenosine monophosphate

carbonyl cyanide m-chlorophenylhydruone

carboxyl

cytochrome c oxidase

cytochrome c oxidase subunit IV dalton

murine dihydrofolate reductase

deoxyribonucleic acid

endoplasrnic reticulum

heat shock protein 70

inner membrane

inner membrane peptidase

intermembrane space

kiloddton

mitochondria1 Dnal homologue

mi toc hondrial GrpE homologue

rnitochondrial heat shock protein 10

mitochondrial heat shock protein 60

rnitochondrial heat shock protein 70

rnitoc hondrial intermediate peptidiw mitoc hondrial processing peptidase

mi tochondrial import stimulation factor

mitoc hondrial deoxyribonucleic acid

matrix-targe ting signal NADH cytochrome bg reductase

Neu rospo ra crassa amino

OM PAGE

PCR

Pi

PO^ pOMD29

RNA

SDS

Tim Tom

TPR

Tx- 1 14

Ydj 1

yTom70p

outer membrane

polyacrylamide gel electrophoresis

polymerase chain reaction

inorganic phosphate preornithine carbarny 1 transferase

hybrid protein containing amino acids 1-29 of yTom70p fused

to DHFR

hybrid protein containing amino acids 1-38 of rat pOCT fused

to amino acids 1 1-29 of yTom70p fused to DHFR ribonucleic acid

sodium dodecy 1 sulfate translocase of the inner membrane

trmslomse of the outer membrane

tetrauicope ptide repeat

Triton X- L 14

yeast homologue of DnaJ yeast Tom70

CHAPTER 1:

Introduction.

1.1 Mitochondria

Mitochondria, are interesting and complex organelles which are located within

eukaryotic cells. They are traditionally viewed as the organelle which contains the

respiratory assembly and the oxidative phosphorylation machinery. However, these are

only two of the many functions which mitochondria perform. They are also involved in the

biosynthesis of pyrimidines, amino acids, phospholipids, nucleotides, folate coenzymes,

heme, and urea (Attardi and Schatz. 1988). As well. the enzymes of the tricarboxylic acid

cycle and fatty acid oxidation are also contained within the mitochondria.

Recently, mitochondria are emerging as a prominent player in apoptosis, or

programmed cell death (Kroemer el cz l . , 1995), with the anti-apoptotic protein, Bcl-2,

located on the outer mitochondria1 membrane (Nguyen et id., 1993). As well, the release

of cytochrome c from the intermembrane space of mitochondria can induce apoptosis (Liu

et id., 1996) and this release can be blocked by Bcl-2 (Kluck et dl . , 1997; Yang rt ol.,

1997). Defects in mitochondrial function have also been implicated in over one hundred

diseases (Luft, 1994), many of which are due to mutations in the mitochondrial DNA

(mtDNA). As well, rntDNA mutations and impaired oxidation occur as secondary

phenomena in aging and age-related diseases such as Parkinson's. Alzheimer's and

Huntington's, amongst others (Luft, 1994). mtDNA is also important in establishing

lineages of animals and the ancestry of humans as shown recently by sequencing mtDNA

from a fossilized Neandertal bone to determine that Neandenals are most likely not the

ancestors of modem humans (Krings et of., 1997).

Mitochondria contain their own double stranded circular DNA (mtDNA), consisting

of 16,569 base pairs in humans (Anderson e t nl., 198 l), which encodes thirteen proteins,

twenty two transfer RNAs and two ribosomal RNAs (for review see Clayton, 1991). The

thirteen proteins are translated within the mitochondrion and encode essential components

of the oxidative phosphorylation system, including key subunits of each of four respiratory

complexes (Wallace, 1986; 1994; Clayton, 1991).

In contrast to the thirteen mitochondrially encoded proteins, the majority

(approximately 1000) of the other mitochondria1 proteins are encoded by the nucleus and

translated in the cytosol. Since mitochondria grow and divide from pre-existing

mitochondria, they have to expand their DNA and proteins within by taking up more

proteins from the cytosol. Mitochondria are composed of two membranes, the outer and

the inner, and therefore delineate four compartments, the two membranes. the

intermembrane space between the two membranes and the matrix within the inner

membrane. Therefore. not only do the cytoplasrnically synthesized proteins need to be

targeted to the mitochondria. they also need to be sorted within the mitochondria to reach

their final location.

1.2 Mitochondria1 Import

1.2a Overview

The specificity of imported proteins into the mitochondria is due to several

interacting factors. These include targeting signals found within the proteins themselves

and the translocation machinery located within the outer and inner membrane of the

mitochondria. As well, import is dependent on several other factors such as, cytosolic

chaperones, the membrane potential (Ay) across the inner membrane, energy (ATP) in

both the cytosol and the matrix and chaperones in the matrix. A summary of the steps

involved in import is as follows (Figure I ) (Reviewed in: Schatz, 1996; Neupen, 1997;

Pfanner and Meijer, 1997). Mitochondrial preproteins are synthesized on ribosomes in the

cytosol and released. They are then transferred to the mitochondria aided by cytosolic

factors (chaperones) which keep the preproteins import competent and prevent misfolding

or aggregation. The preproteins are then recognized through targeting signals within the

preprotein sequence which interact with receptors on the outer membrane (OM) and

possibly with the lipid bilayer itself. The preproteins which are interacting with the surface

recep ton are then transferred to the &amlocase of the outer mi toc hondrial membrane (Tom)

Figure 1. Protein import into mitochondria. An overview of the components

involved in protein import. Chaperones assist the preprotein in the cytosol. mitochondria1

import stimulation factor (MSF) and Hsp70/Ydj 1. The preprotein then interacts with

components of the outer membrane translocation machinery, the receptors Tom70/Tom37,

Tom20/Tom22 and the pore components Toms, Tomb. Torn7 and Tom40. If the

preprotein is inserting into or passing through the inner membrane it then interacts with the

components of the inner membrane translocation machinery, the pore components, Tim44,

Tim23 and Tim17 or with other components Tim22 and Tim1 L. The preprotein is

translocated across the inner membrane by the action of Tim44 and mHsp70 in the matrix.

The chaperone mHsp70 is assisted by its cofactors mGrpE and Mdj 1. mHsp60/mHsp 10

aid in the proper folding and oligomerization of the translocated proteins. Matrix

peptidases cleave off the matrix-targeting signal, a- and P-MPP and MIP. Inner membrane

pepridases, IMP-1 and IMP-2, cleave off bipartite sorting signals. Ay across the inner

membrane is needed for translocation into or across the inner membrane, and is negative

inside the matrix. Cyto, cytosol; OM, outer membrane; IMS, intermembrane space; IM,

inner membrane. See text for details.

ATP

Cyto

OM

IMS

IM

ADP

Matrix

mGrpE ATP

1 1-y ADP

(See Pfanner et d l . , 1996, for nomenclature) and are either inserted into the OM,

translocated into the intermembrane space (IMS) or they then interact with the surface of the

inner membrane (IM). The preproteins then insert into the ganslocase of the inner

mitochondria1 membrane (Tim) (Pfanner et al., 1996) in a A y dependent manner. The

preproteins which are translocated through the OM and IM are aided by the mitochondrial

heat shock protein 70 (mHsp70)-Tim44 complex which is located within the matrix and is

ATP dependent. Cleavable presequences are cleaved in the matrix by the mitochondria1

processing peptidase (MPP) and the proteins are either inserted into the IM or trmslocated

completely through to the matrix. IMS proteins are also cleaved on the IMS of the IM by

the inner membrane peptidase (IMP) and released. Inside the matrix the folding of the

proteins is assisted by the chaperones mHsp7O and mitochondrial heat shock protein 60

(rnHsp60) and other co-chaperones. These are the general steps of import, as well, some

proteins are thought to insert into the IM or translocate to the IMS by export from the

matrix after complete translocation (conservative sorting) and cytochrome c can cross the

OM without the use of the Tom complex.

1.2b Cytosolic Chaperones

After synthesis, mitochondrial preproteins in the cytosol need to be directed to the

mitochondria in a relatively unfolded state suitable for translocation. Also, the proteins

have to be kept in an unaggregated state and be protected from degradation. This is

accomplished through interactions with chaperones. Chaperones are proteins which can

bind to and stabilize other proteins, in order to ensure that subsequent interactions can

occur correctly, such as folding, oligomeric assembly, transport to a subcellular component

or degradation (Had , 1996; Rassow rt a/., 1997).

Several chaperones have been identified as being involved in mitochondrial

targeting (Hohfeld and H a d , 1994; Mihara and Omun, 1996). Some of these have not

been purified, but were identified in reticulocyte lysate (Argan et al., 1983; Miura et al.,

1983; Pfanner and Neupert, 1987; Sheffield et nl., 1990). Another which was partially

purified from yeast cytosol, is a stimulation factor with an apparent molecular mass of -40

kDa (Ohta and Schatz, 1984). hsp70 (Ssalp md Ssa2p in yeast) (Rassow et al., 1997) is

involved in mitochondrial protein import as well as in protein import into the endoplasmic

reticulum (ER) (Chirico et al., 1988; Deshaies et al., 1988; Murakarni et al., 1988). Hsp70

is a general factor which binds to stretches of large hydrophobic and aromatic amino acid

residues (Fourie et dl.. 1994). It is not a specific chaperone for mitochondrial import.

although it can also bind to some mitochondrial presequences (Endo et al . , 1996).

However, Ydj I . a yeast homologue of Esherishia coli Dna J (Cyr er al., 1994) has been

suggested to be responsible for the targeting function of Hsp70 for import into

mitochondria (Caplan and Douglas, 199 1: Atencio and Yaffe, 1992; Caplan r t al., 1992a).

perhaps through farnesylation of Ydj I which will attach it to a membrane (Caplan rt nl.,

l992b). More specific factors for mitochondrial import which actually recognize the

matrix-targeting signal (MTS) of mitochondrial precursor proteins and which have been

purified from reticulocyte lysate using affinity chromatography with a MTS are "targeting

factor", which has an apparent molecular mass of -28 kDa (Ono and Tuboi 1988; 1990),

and "presequence-binding factor", which has an apparent molecular mass of -50 kDa.

Presequence-binding factor is also present in the cytosol of rat heart and liver (Murakarni

and Mori, 1990).

A factor has also been purified from rat liver cytosol using affinity chromatography

with a MTS and it is named "&xhondrial import stimulation factor" (MSF). It consists

of two subunits with apparent molecular masses of 30 and 32 kDa (Hachiya et al., 1993).

It was cloned and shown to be a member of the family of 14-3-3 proteins (Alam et al.,

L994). MSF recognizes the MTS and targets the proteins to the surface of the mitochondria

where they interact with the OM receptors (Hachiya et a!., 1994; Komiya et al., 1994;

Hachiya et a!., 1995; Komiya et al., 1996). MSF will stimulate the import of many

mitochondrial precursor proteins including some which do not have a MTS, such as porin.

It is also able to unfold, in an ATP-dependent, manner mitochondria1 precursor proteins

which have been previously synthesized and aggregated in wheat germ lysate.

Mitochondria1 precursor proteins when bound to MSF induce its ATPase activity (Komiya

et al., 1994). Therefore, MSF has dual functions, presequence recognition and ATP-

dependent unfolding of proteins.

Two pathways have been suggested for targeting to mitochondria dependent on the

chaperone involved. MSF or Hsp70. The Hsp70 pathway is ATP independent. N-ethyl

maleimide insensitive and uses the receptor subcomplex of Tom20-Tom22. This

subcomplex is also used by unfolded proteins e.g. urea denatured proteins (Hachiya et nl.,

1905). The second pathway involving MSF targeting, is ATP-dependent, N-ethyl

rnaleimide sensitive and uses the receptor subcomplex Tom70-Tom37. After release of

MSF, the precursor protein is then transferred from Tom70-Tom37 to the Tom20-Tom22

subcomplex (Hachiya et al., 1995).

1 . 2 ~ Translocase of the Outer Mitochondria1 Membrane (Tom)

The OM and LM each contain translocation machinery (Tom and Tim), respectively,

which can act concertedly to import proteins into or across the IM or which can act

independently of each other. Most of the components of Tom and Tim have been identified

in yeast and Nerlrospora crassa (N. crassa). The mammalian homologues are just

beginning to be identified. Earlier reports showed Tom containing at least seven protein

components (Moczko rt ol., 1992; Sollner rt al., 1992). Now the number of components

is up to at least nine.

The Tom is composed of both receptor proteins and general insertion pore proteins.

The components are not stable protein complexes, however, the components are organized

such that they dynamically interact in subcomplexes. There are four receptor proteins,

Tom70 (Tom72), Tom37, Tom22, and Tom20, three small proteins, Tom5, Tom6 and

Tom7, and the main constituent of the import pore, Tom40. The OM receptors have been

shown to exist in at least two large complexes, one of 400 kDa which contains Tom22.

Tom5 and Tom40 (Dekker et al., 1996; Dietmeier et al., 1997) and one of 120 kDa to

which Tom70 belongs (Dekker et al., 1996).

Tom70, Tom72 and Tom20 all have a NH2-terminal signal-anchor sequence with a

cytosolic hydrophilic region. Tom22 has the opposite orientation to Tom70 and Tom20,

with an NH2-terminal cytosolic region. Tom72 (also called Turn7 1), which has been

identified in yeast. is a close relative of Tom70 with 49% identity and 70% similarity

(Bomer rr crl., 1996: Schlossmann et ul., 1996; Haucke and Lithgow, 1997) however, it is

expressed at a low level and does not have a major role in mitochondria1 biogenesis.

Tom70 is targeted to and anchored in the OM of mitochondria by its NH2-terminal

hydrophobic domain, with the bulk of the protein in the cytosol. It was shown to be an

import receptor for some but not all mitochondria1 precursor proteins (Hines et dl., 1990;

Sollner et d., 1990). Tom70 can form homodimers (Sollner et al., 1992) as well as

heterodimers (Shore et cil., 1995) with the transmembrane domain contributing to dirner

formation (Millar and Shore, 1993; 1994). The hydrophilic cytosolic domain has been

shown to interact in a specific manner with precursor proteins (Schlossmann et nL, 1994).

Torn37 has only been identified in yeast and is predicted to contain two

transmembrane regions. It has been proposed to form a subcomplex with Tom70 (Gratzer

et al., 1995).

Tom20 was identified in N. crassa and yeast (Sollner, et nl., 1989; Ramage rt al.,

1993) and more recently in humans (Goping et al., 1995; Seki et a!., 1995; Hanson et of.,

1996) and rat (Iwahashi et ai., 1997) as a rnitochondrial OM import receptor. It has a short

NH~-terminus in the IMS and a large hydrophilic cytosolic region. Three characteristic

structural features have been identified, a NH2-terminal hydrophobic segment, a putative

tetmtricopeptide repeat (TPR) motif in the middle and a COOH-terminal negatively charged

segment. The TPR motif in human Tom20 only shows weak homology to the B-domain

(Goping et al., 1995). How Torn20 interacts and recognizes precursor proteins is being

investigated. It was shown that electrostatic interactions are involved between the

negatively charged COOH-terminal region of Tom20 and positively charged presequences

(Haucke et d., 1995). In binding studies, Schleiff et al. showed that in addition to binding

precursor proteins with positively charged arnphiphilic presequencrs, Tom20 interacts with

other types of precursor proteins as well. Different regions within Tom20 seem to be

involved in the recognition of the different classes of precursor proteins (Sc hlriff rt a!.,

1997a; 1997b).

To date, Tom22 is the only essential subunit of the protein import receptors

(Nargang rr id., 1995). It contains negatively charged amino acids, the "acid bristle", and a

45 amino acid IMS region which also contains negatively charged amino acids (Kiebler cr

trl., 1993: Lithgow et d . , 1994; Honlingcr et al., 1995; Nakai and Endo, 1995; Nargang er

trl., 1995). There are conflicting reports regarding the role of the MS region of Tom22.

This region was thought to play a role in the binding of the mitochondria1 targeting signal

during protein import into the mitochondria. The IMS region was deleted and it was found

that the import of certain precursors was decreased by 3- to %fold (Bolliger er al., 1995).

Although other groups concluded that the IMS region of Tom22 does not play a crucial role

in the import of preproteins into mitochondria (Nakai et nl., 1995; Coun er dl., 1996),

however, it enhanced the efficiency of transferring the preproteins tiom the trms side of the

OM to the Tim (Court er ul., 1996).

Tom20 and Tom22 probably function as a unit (Mayer er ol., 1995) presenting their

acid bristle domains as a binding site for the precursor targeting signal since mutations to

these regions impairs import into the mitochondria, in part due to lower productive binding

of the precursors (Bolliger et al.. 1995).

To summarize, the receptor complexes function to capture preproteins from the

cytosol via at least two different pathways. The f ia t pathway is direct interaction of the

proteins with a positively charged presequence with Tom20-Tom22 which has negative

clusters. Also, proteins which use the chaperone Hsp70 in the cytosoi, seem to then

interact with the Tom20-Tom22 subcomplex. The second pathway is used by hydrophobic

proteins and ones which have internal targeting sequences. This pathway is also favored

by preproteins which interact with the chaperone MSF. In this pathway, the preprotein

interacts with Tom70(72)-Tom37 initially and is subsequently passed on to the Tom20-

Tom22 subcomplex. Some preproteins can use both subcomplexes and there does seem to

be some contact between subcomplexes through Tom20 and Tom70.

The receptors contain and are proposed to interact with one another throush TPR

motifs. TPR motifs are found in functionally diverse proteins and are thought to mediate

protein-protein interactions (Boguski et a le , 1990; Goebl and Yanagida, 199 1 ; Lamb er d..

1995). Tom70 has seven TPR motifs (Ramage et crl . , 1993) and Tom37 (Gratzer er al.,

1995) and Torn20 each have one (Moczko et al., 1994). The two receptor subcomplexes

interact with one another through the Tom70 and Tom20 subunits and 3 mutation of the

TPR motif in Torn20 abolishes this association. When the association is abolished, import

of precursors which use the Tom70-Tom37 subcomplex first followed by Tom22-Tom20

subcomplex is inhibited (Haucke et ul., 1996).

There are three small proteins associated with Tom; these are Toms, Tom6, and

Torn7. Tom5 has n COOH-terminal anchor which is proposed to be in the membrane,

flanked by positive charges and a net negatively charged NH2-terminal. It has been shown

to be the next protein to interact with the preprotein after the Tom20-Tom22 subcomplex

and before Tom40 (Dietmeier et al., 1997). Tom6 seems to promote the association of

Tom70-Tom37 and Torn20-Tom22 with Tom4O. If Tom6 is deleted then the transfer of the

translocating preprotein from the receptors to the pore is delayed (Alconada er dl., 1995;

Cao and Douglas, 1995). Tom6 also has a COOH-terminal anchor which is responsible

for targeting Tom6 to the mitochondria (Kassmbrock er a!., 1993). Tom7 promotes the

dissociation of Tom20-Torn22 from Tom40 and Tom20 from Tom22, i.e., the opposite

function to Tom6. Tom7 has been proposed to play a role in the sorting and accumulation

of preproteins at the OM. Without Tom7, there is an increase in the stability of association

between Tom20-Tom22 and Tom4O. The sorting of porin is affected by the lack of Tom7

(Honlinger et al., 1996). Therefore, the dissociation of the translocase subunits is

important for the lateral release of the preprotein into the lipid phase of the OM.

Tom40 was first identified as part of the insertion pore through crosslinking with

precursor proteins (Vestweber et al., 1989). It was then cloned in yeast and N. Crnssa

(Baker et nl., 1990; Kiebler et al., 1990). Recently it has been proposed that Tom40 forms

the "tmns site" of binding (a site of binding for the preprotein on the IMS side of the OM)

for precursor proteins during import into the mitochondria. This was shown through

crosslinking of the presequences of precursor proteins. The presequence is in contact with

Tom40 early during translocation and after losing contact with TodO/Torn22, it is still in

contact with Tom40 (Rapaport, et al., 1997). Tom40 is embedded in the OM, but the

structure of the translocation pore is not known.

1.2d Translocase of the Inner Mitochondria1 Membrane (Tim)

The Tim machinery is unique and independent from the Tom machinery. It has

been shown using mitoplasts. in the absence of an OM, the IM can still translocate

preproteins (Ohba and Schatz, 1987; Hwang et a/ . , 1989: Li and Shore, 1992b).

Therefore, the Tim must be able to recognize import signals and translocate preproteins on

its own. To date five subunits of Tim have been identified and cloned and as well, four

other proteins have been found to be associated with Tim.

Tim17 is an essential IM protein, it does not have a typical MTS but instead, its

NH2-terminal region has a net negative charge. Tim17 is predicted to span the IM up to

four times and it is in close proximity to the protein import site (Kiibrich et al., 1994;

Maarse et al., 1994; Ryan et al., 1994).

Tim23, which was cloned in yeast (Dekker et al., 1993; Emtage and lensen, 1993),

is also an essential IM protein with up to four transmembrane regions in its COOH-terminal

half and a very high number of acidic amino acid residues in its NH2-terminal half. The

NH2-terminus is not cleaved upon import although A y is needed for its import. The NH2

and COOH-terminus of Tim23 are in the IMS and the hydrophobic regions of Tim23 show

homology to those of Timl7. Tim23 forms dimers whose formation are promoted by Av.

The dimers are formed through interactions in the second half of the NH2-terminal region

of Tim23 which contains a heptad leucine zipper motif. Upon presequence binding the

dimer dissociates and during preprotein translocation, monomeric Tim23 is present (Bauer

et al., 1996). The Tim 23 dimers are proposed to act as a receptor on the surface of the LV

and its dissociation triggers the opening of the Tim channel allowing insertion of the

preprotein wtule preventing leakage of ions across the membrane.

Tim17 and Tim23 have similar orientations in the membrane and are thought to

form part or ail of the channel for preproteins. They have been shown to be present in a 90

kDa complex by blue native electrophoresis. The complex is thought to contain two copies

of each of Tim17 and Tim23. During preprotein translocation of proteins with a cleavable

presequence, the 90 kDa complex becomes a 600 kDa complex linking the Tim machinery

with the Tom machinery 400 kDa complex. A single mutation in the membrane spanning

segment of Tim23 will destabilize the 90 kDa complex and inhibit the import of preproteins

with cleavable MTS's (Dekker et ai., 1997).

Tim44 is the third essential protein of the Tim machimy. Unlike Tim17 and

Tim23 it is a peripheral membrane protein with its bulk on the matrix side of the IM, and

although it has no potential hydrophobic transmembrane regions, its COOH-terminus was

shown to be on the IMS side of the IM (Maarse et al., 1992; Scherer et al., 1992). As

well, Tim44 is synthesized with a cleavable MTS at the NH2-terminus (Blorn rt al., 1993).

In addition to the Tim17-Tim23 subcornplexes, Tim23-Tim44 subcomplexes have also

been identified (Borner et al., 1997). Tim44 is also found in a 1: 1 complex with mHsp70

(Kronidou et nl., 1994; Rassow et al., 1994; Schneider et a[., 1994). Tim23 may link

Tim44-mHsp70 to the IM pore, since Tim23 is found in two pools, one with Tim17 and

one with Tim44 (See more about the Timu-mtHsp70 complex in the section entitled

Matrix Chaperones below).

Two other members of Tim have been identified and cloned although they do not

seem to be located in the preprotein translocation pore for preproteins with cleavable

presequences. Timl I is a non-essential protein and has a hydrophobic NHz-terminus and

a hydrophilic COOH-terminus which is most likely in the IMS. Timl I is involved in the

import of IMS precursors (e .g . cytochrome b?) and can be found closely apposed to the

membrane anchor sorting signal (Tokatlidis et ai., 1996). Tim22 is an integral membrane

protein which is essential for viability. The second half of the protein has some sequence

homology to Tim17 and Tim23 and although it is found in a complex of 300 kDa it is not in

association with the other Tim components, Tim 17 and Tim23. It functions in the import

of proteins of the IM such as the ATP/ADP carrier family and others proteins without a

presequence (Sinenberg et al., 1996).

Two other reports each have shown that two different proteins are in a complex

with Tim17 and Tim23 but these proteins have not been further identified or cloned. One

reports proteins of 20 kDa and 55 kDa (Blom et of., 1995) while the other reports proteins

of 14 kDa and 33 kDa (Berthold et a/ . , 1995).

The components of the Tim are just beginning to be elucidated and their interactions

and roles in the translocation of different types of preproteins are starting to be understood.

However, there is still much to be determined regarding this machinery.

1.2e Matrix Chaperones

Protein transport into and across the IM requires ATP within the matrix. This

requirement for ATP correlates with the requirement for mHsp70. mHsp70 is the

mitochondrial homologue of the Hsp70 family of chaperones which prevent aggregation

and improper folding of proteins. Hsp70 cycles on and off the substrate proteins coupled

to cycles of ATP binding and hydrolysis. Two binding regions have been identified in

Hsp70 proteins, the NH2-terminal region binds adenine nucleotides and the COOH-

terminal region binds short stretches (-7 amino acids) of extended polypeptides. When

Hsp70 is complexed with ADP, it is bound tightly to the extended polypeptide. Whereas

upon ATP binding. the polypeptide is released (Glick 1995). mHsp70 is an essential

protein for viability. Mitochondria with a temperature sensitive mutation in mHsp70 have

defects in the import of preproteins (Kang et nl., 1990; Gambill rt al., 1993).

Translocating precursor proteins are associated with mHsp7O in the matrix (Ostermann et

al., 1990; Scherer et al., 1990).

Tim44 forms a subcomplex with mHsp70 and mitochondria1 GrpE (rnGrpE) which

is also bound to the translocating preprotein (Kronidou et al., 1994). 1045% of rnHs~70

is bound to T i m 4 at the matrix side of the IM (Kronidou et nl., 1994; Rclssow et al., 1994;

Schneider et trl., 1994). This subcomplex uses ATP and is thought to be the core of an

ATP driven import motor. How the motor works in aiding translocation across the IM is

not completely understood and there are two models which have been proposed. The first

is the "Brownian rachet" model while the second is the "translocation motor" model. The

first model involves the trapping of the preprotein while the second involves pulling the

precursor. These models have to explain both the translocation of the protein into the

mitochondria and the unfolding of the preprotein on the surface of the mitochondrion.

These so-called "translocase" and "unfoldase" activities have been examined by several

different groups and are starting to be sorted out. In the Brownian ratchet model, the

protein randomly vibrates due to Brownian motion in the translocation pore. Inside the

matrix, a molecule of mHsp70 binds a section of the precursor and traps it, preventing it

from moving back out of the pore. After a series of binding events, the precursor would be

imported. The model for the translocation motor driven by mHsp70 is as follows (Horst et

al., 1997). mHsp70-ATP associates weakly with Tim44 and incoming precursor proteins,

after ATP hydrolysis mHsp70-ADP binds tightly to Tim44 and the precursor. Then

mHsp70ADP undergoes a conformational change to pull a segment of the preprotein into

the matrix. mGrpE then binds to promote ADP release, after which ATP rebinds to

displace mGrpE and release rnHsp70 from Tim44 and the precursor. This sequence cycles

until the preprotein is imported. It is possible that the import of preproteins into the

mitochondria involves the Brownian rachet model for the initial translocation of the MTS,

until mHsp70 can bind and thereafter, the translocase motor will be involved with

unfolding and translocation of the protein.

The Hsp70 homologue in bacteria. DnaK. functions in association with GrpE and

DnaJ. GrpE is a nucleotide exchange factor and functions to accelerate the release of

DnaK-bound nucleotide, to allow DnaK to recycle more efficiently (Liberek et nl., 199 1).

DnaJ increases the rate of ATP hydrolysis, stimulating the ATPasc activity. Mitochondria1

homologues of these two proteins have been found to exist in the matrix. mGrpE was

purified in association with mHsp70 and subsequently cloned from yeast (Bolliger rt al.,

1994: krda et al., 1994; Laloraya et dl . , 1994). Therefore rnGrpE could have a function in

the translocation of precursors or in the refolding of precursors in the matrix. Recently it

was shown that mGrpE functions at several steps of the translocation pathway. It aids in

the formation of a complex between Tim44 and mHsp70 in the presence of ATP, after ATP

hydrolysis and Pi release, rnGrpE aids the dissociation of the mHsp7O-ADP from the

T i m 4 complex and as well it promotes the release of ADP form mHsp70 and the uptake

of ATP by mHsp70 (Schneider et al., 1996). Mitochondria1 DnaJ (Mdj 1 ) is associated

with the IM, in the matrix. It is not lethal if deleted, but the folding of newly imported

proteins was reduced. Therefore, it makes the folding process of newly imported proteins

more efficient (Rowley et al., 1994).

As indicated above, once inside the matrix, translocated proteins need to be folded

to their native state. After import they are transferred from mHsp7O to the chaperonin

rnHsp60, possibly aided by Mdj 1 (Reading et al., 1989; Cheng et al., 1989). Temperature

sensitive mutants of mHsp60 will accumulate imported proteins as insoluble aggregates,

therefore it functions in the refolding and oligomerization of some imported proteins

(Cheng et al., 1989). rnHsp60 is assisted by rnHsplO in the oligomeric assembly and

refolding of the proteins in an ATP dependent manner (Rospert et al., 1993a; 1993b).

Both rnHsp6O and mHsp 10 are essential in yeast and they are homolognus to GroEVGroEs

in bacteria. However, not all proteins need the assistance of mHsp6O and mHsp10 to attain

their native state (Rospen et nl., 1996). mHsplO is also involved in keeping proteins

which are sorted conservatively in a conformation competent for sorting back through the

1-M after having reached the matrix (Hohfeld and Hartl. 1994a).

1.2f Energetics

The import of preproteins into mitochondria requires energy at different steps.

(Beasley et al., 1992). Depending on the final destination of the preprotein, ATP is needed

in the cytosol andor in the matrix. As well, for preproteins which are translocating into or

across the inner membrane, there is a requirement for the Ay across the IM (negative inside

the matrix).

1.2f-1 ATP Requirement

There are three steps of protein import which require ATP. One at the cytosolic

side of the OM and two more are within the matrix. The requirement for ATP outside the

OM is most likely needed to keep the precursor proteins import competent, that is, in a

loosely folded state through interactions with chaperones such as Hsp7O and MSF. The

requirement for ATP in the cytosol can be bypassed by denaturing the preproteins with urea

(Pfanner et al., 1988; 1990). As well, ATP hydrolysis is needed for the release of the

preproteins from the chaperones as they are transferred to the OM import receptors. (See

section entitled Cytosolic Chaperones).

ATP is needed inside the matrix for preproteins to translocate into or across the IM

(Hwang and Schatz, 1989). mHsp70 needs ATP as it binds to the translocating preprotein

and pulls the preprotein across the IM through cycles of binding and release. A second

reaction within the matrix which requires ATP is catalyzed by mHsp60. mHsp60 interacts

with proteins after they are released from mHsp70 and aids in their folding or

oligomerization (Ostermann et al., 1989; Manning-Krieg et al., 199 1). (See section entitled

Matrix Chaperones).

1.2f-2 L\y Requirement

Ay! is needed to import preproteins into or across the LM. Once the presequence is

in the matrix, Ay is no longer needed for the import of the rest of the preprotein. A v could

function to either influence the MTS electrophoretically or by activating an LM protein,

either through binding of the preprotein or by opening a pore. There is evidence to support

both of these possibilities. It was shown that the insertion of the positively charged

presequence across the IM was by an electrophoretic effect since different thresholds of

inhibition were found for different MTS 's (Martin rt ol.. 1991) and since hy is only

needed when the presequence contains positively charged amino acids (McBride cr (11..

1995). However, this doesn't rule out the possibility that the threshold for opening a

channel is below the level needed to drive the movement of the presequence. Indeed,

Tim23 has been shown to form dimers in the presence of Ay which dissociates in the

presence of a MTS (Bauer et al., 1996). Therefore, it appears that a combination of both

gating and electrophoretic effects is involved.

1.2g Processing Proteases

Processing enzymes are present within the mitochondria to cleave signal sequences

during or after import. In the matrix, the metal dependent endopeptidase, MPP consists of

a- and p- subunits (Reviewed in: Brunner et al., 1994; Luciano and GCli, 1996). Both of

the subunits are needed for activity, a-MPP is thought to bind the preprotein while the

catalytic activity resides with p-MPP. Both subunits are also essential for viability in yeast.

Sometimes the MPP cleaved proteins undergo a second processing event in the mauix.

This is accomplished by mitochondria1 intermediate peptidase (MIP) which is also located

in the matrix (Reviewed in: Pratje et al., 1994). MIP removes an octapeptide from some

NH2-termini which are generated by MPP.

The IM also contains a proteolytic activity on the IMS side. The inner membrane

peptidase (IMP) consists of two subunits (IMP1 and IMP3 which have different substrate

specificity (Nunnari et d., 1993; Reviewed in: Isaya and Kalousek, L99.I). The

polypeptides are membrane bound and the active sites are in the IMS. For proteins which

contain bipartite signals (see section on Topogenic Sequences), after the initial cleavage

in the matrix by MPP, the second part of their sorting signal is cleaved in the IMS by IMP.

1.3 Topogenic Import Signals

In order to function properly, the cell needs to ensure that newly synthesized

proteins are targeted to their correct location. In order for proteins to be targeted, topogenic

signals are fcund within the proteins themselves, often as a NHz-terminal extension. Most

signals consist of either a primary consensus sequence or as a secondary structural feature

of the protein. Proteins targeted to the nucleus and most of the proteins targeted to

peroxisomes contain primary consensus sequences. The peroxisomal PTS 1 signal usually

consists of the sequence SKL, or a variant thereof, at the COOH-terminus of the protein

(Rachubinski and Subramani, 1995). Nuclear localization signals are highly enriched in

basic amino acids and consist of either a single or bipartite signal. One example is

PKKKRKV, which is the nuclear localization signal of the SV40 large T antigen (Silver,

199 1; Gerace, 1995). In the absence of a primary sequence, targeting signals that exist

within the secondary structure often consist as an a-helix. This is true for proteins which

are targeted to the ER, the mitochondria and some of the proteins targeted to the

peroxisomes. The signal for ER targeting is NH2-terminal consisting of 15-30 amino

acids, the NH2-terminus has a net charge of +2, followed by a 7-15 amino acid

hydrophobic core, and then by a 5 amino acid polar region (Lazdunski and Benedetti,

1990; Rusch and Kendall, 1995). The hydrophobic region either inserts the protein into

the lipid bilayer or it is cleaved during translocation releasing the mature soluble protein into

the lumen of the ER. Peroxisomal PTS2 signals are located at the NH2-terminus and

consist of about 26-36 amino acids which are cleaved after import (McNew and Goodman,

1996). They resemble the MTS for mitochondria1 targeting (see section on Matrix-

Targeting Signals) and a disease condition exists where a peroxisomal protein is

mistarge ted to the mitochondria (Dan pure, 1995).

Proteins destined for the mitochondria contain either a cleavable presequence on the

NH2-terminus, a signal within the mature protein or a combination of types of signals.

These signals contain sufficient information to target the protein to the final localization

within the mitochondria. Since fusion proteins containing the targeting signal joined to a

cytosolic precursor protein such as dihydrofolnte reductasr (DHFR) are still targeted to the

correct location within mitochondria.

1.3a Matrix-Targeting Signals

The best characterized mitochondriill signal is the NH2-terminal matrix-targeting

signal (MTS). MTS are about 20-60 amino acids in length and lack any homologous

amino acid sequence. They have a high net positive charge due to a high content of

arginine and lysine residues and a lack of negatively charged amino acids, they contain

frequent hydroxylated residues and have the ability to form an amphiphilic a-helix with a

positively-charged face and an apolar face upon binding to a membrane surface (Epand et

al., 1986; von Heijne 1986; 1990; Roise and Schaa, 1988). Once inside the matrix most

MTS's are cleaved by MPP. The MTS by itself can inhibit import of authentic

mitochondrial precursor proteins showing that they interact with components of the

translocation machinery (Gillespie et al., 1985).

1.3b Outer Membrane Signals

The OM of mitochondria contains 8-10% of the total protein within the organelle,

including components of Tom, enzymes and pore structures. The OM signals are not

cleavable like the MTS and reside within the mature protein sequence. There are two

classes of integral OM proteins. One class of proteins within the OM are those which form

a P-barrel structure such as porin and Tom4O. The topogenic features needed for the

import of P-barrel proteins have not been determined. however. for porin some signals

have been defined and they reside within both the NHz- and COOH-termini (Hamajirna et

d . , 1988). Specifically, certain residues within the COOH-terminus were found to be

important for insertion (Smith ef nl., 1995).

Another group of proteins which exist in the OM are those which contain a single

hydrophobic region, predicted to be a-helical and bitopic, spanning the membrane once.

One such protein which has been studied in detail is Tom70 (yMas70p, MOM 72) (Hase e t

dl 1984; Hurt et a/. , 1985; Nakai et al., 1989; Li and Shore, 19921; b; McBride et al.,

1992; Steenaart et d., 1996). The topogenic information of Tom70 resides within amino

acids 1-29, this region functions to both target and insert the protein into the OM with an

Nin-Gout orientation. Amino acids 1- 10 are positively charged and hydrophilic while

amino acids 11-29 are hydrophobic and are predicted to be the transmembrane segment.

delineating two regions. A fusion protein consisting of amino acids 1-29 fused to DHFR.

will target to the OM and be inserted with the correct orientation (Nin-Gout). The

topogenic information has been examined and it was determined that amino acids 11-29

contain all the information needed to target and insert the fusion protein, while the

hydrophilic region serves to enhance the rate of import of the protein (McBride et al.,

1992). The hydrophilic region itself has very little targeting information in mammalian

systems, although it can function as a MTS in yeast (Hurt et al., 1985; McBride et al.,

1996). Since the same sequence which targets and initiates translocation of the protein,

also abrogates the translocation and anchors the protein in the lipid bilayer the targeting

domain is referred to as a signal-anchor domain (McBride et al., 1992, Li and Shore,

1992a). When the hydrophilic region of the Tom70 signal-anchor is replaced by a MTS

from preornithine carbamyl transferase (pOCT) such that it is contiguous to the

transmembrane region of the signal-anchor, this longer signal-anchor will still arrest the

protein in the OM, although it is now inserted with the opposite orientation (Gin) (Li and

Shore, 1992a). A signal-anchor sequence can also be created by inserting a heterologous

stop-transfer sequence (the membrane anchor sequence of the vesicular stomatitus virus

glycoprotein) contiguous to the pOCT MTS which also arrests the protein in the OM with

its COOH-terminus inside (Nguyen et nl., 1988). This reversal of orientation in the OM is

due to the amphiphilic character of the retention sequence rather than the number and

position of positively charged amino acids. As well this amphiphilic positively-charged

sequence has lipid-binding characteristics implicating the membrane surface as a

contributing factor in retaining the NH2-terminus on the cytosolic side of the membrane

(Steenanrt rt c z l . , 1996; See Chapter 2). The signal-anchor domain of Tom70 also has the

ability to homodimerize (Millar and Shore, 1993; 1994) and heterodimerize (Shore et al.,

1995) which may play a role in its function as a receptor for protein import into

mitochondria. Tom20, another OM protein import receptor, also contains an NH2-terminal

signal-anchor as seen by the ability of the NHz-terminal28 amino acids to target and insert

DHFR into the OM (Goping et al., 1995). Signal-anchor sequences can also exist at the

COOH-termini of proteins such as Bcl-2. The sequence is found within the COOH-

terminal 22 amino acids (Nguyen et al., 1993) which inserts the protein into the OM with

an Ncyto-Gin orientation. Agin a fusion protein consisting of DHFR fused to the COOH-

terminal 22 amino acids will target and insert into the OM with the correct orientation

(Nguyen et ul., 1993). Tom6 also contains a COOH-terminal signal-anchor (Cao and

Douglas, 1995). Therefore, signal-anchors can function at either the NH2- or COOH-

termini of proteins.

Although signal-anchors seem to be the method of choice for insertion of bitopic

OM proteins, how the OM receptors recognize the signals and insert the proteins, remains

to be de terrnined.

1 . 3 ~ Inner Membrane and Intermembrane Space Signals

The IM contains a diverse set of proteins including the proteins required for

oxidative phosphorylation and nucleotide and substrate transport. These proteins have

many different topological arrangements in the membrane with one or more transmembrane

domains. Some of these proteins are encoded by mtDNA, while the bulk are encoded by

the nuclear genome.

The topogenic signals for bitopic proteins of the IM consist of a cleavable MTS and

a hydrophobic sorting signal downstream. This hydrophobic region can act as a stop-

transfer signal in the IM for proteins such as cytochrome c oxidase subunit Va (Glaser ct

dl., 1 !WO), stopping tnnslocation, inserting the protein into the membrane and becoming

the transmembrane domain. A protein which targets to the IM with an Nin orientation can

also be constructed by placing a heterologous stop transfer sequence downstream of a MTS

(Nguyen and Shore, 1987). The MTS is cleaved in the matrix by MPP, generating the

mature protein. It is not known what factors determine whether a hydrophobic region will

act as a signal-anchor, a stop-transfer or a transfer-arrest sequence. This phenomena was

investigated and it was found that in order for a hydrophobic region to function as a stop-

transfer region in the IM, when it is located downstream of a MTS, it needs to have a fairly

high hydrophobicity (Steenaart and Shore, 1997a). The results of this study are presented

in Chapter 3.

A second group of IM proteins are the multiple membrane spanning proteins. This

group consists of proteins such as uncoupling protein, the ATPIADP carrier and the Pi

carrier. They each contain six transmembrane regions and have an Nims-Cims orientation.

The targeting signals of these proteins are located within the mature protein and they do not

contain a cleavable NH2-terminal sequence, but generally, it is not known what the signals

are. The first two transmembrane regions of the uncoupling protein were found to be

essential for targeting and insertion into the IM (Liu et al., 1988). The mechanism of

insertion into the IM is proposed to be one of sequential insertion of paired membrane

sequences (Shore et al., 1992).

The targeting signals for proteins of the [ M S are bipartite signals consisting of a

cleavable MTS immediately followed by a hydrophobic portion. The hydrophobic region

is thought to mest translocation without insertion in the LM. The MTS is cleaved off in the

matrix generating an intermediate form. A second cleavage event then takes place on the

IMS side of the IM generating the mature protein which is soluble and released into the

[MS.

Two sorting pathways are proposed to exist for proteins of the IM and the IMS, the

"unidirectional" or stop-transfer method where the protein fiat crosses the OM and then

inserts directly into the IM (Glick rt nl., 19923) or the "conservative" sorting pathway

(Hart1 and Neupert, 1990), in which the proteins are completely translocated through to the

matrix where part of the presequence is cleaved off exposing the sorting signal which then

directs the protein back out of the matrix across the M, using conserved bacterial export

machinery. The conservative sorting pathway is controversial for the proteins cytochromr

c 1 and b2 of the IMS which have also been proposed to be imported by the stop-transfer

method (Glick et al., 1992). This discussion continues to be debated (GWner er al., 1995;

Gruhler er al., 1995).

Recently the insertion of IM proteins from the matrix side has begun to be examined

for both nuclear and mitochondria1 encoded proteins (Hermann et a[., 1995; 1997; Rojo rt

al., 1995). However, very little is known about the targeting signals and the mechanism

and machinery of insertion. The protein components involved in conservatively sorted

proteins or of the proteins synthesized in the matrix of mitochondria have not yet been

identified. Recently, the genomic sequence of yeast Saccharomyces cerevisiar was

searched and it was determined that mitochondria lack any bacterial-type Sec machinery

(Glick and von Heijne, 1996). It was postulated that these proteins may be translocated by

a Sec-independent pathway.

It is also not known how the hydrophobic transmembrane regions of proteins of the

IM avoid insertion into the OM and are able to tnnslocate through it. One theory is that

once the Tim is engaged by the MTS, the Tom is no longer able to recognize and insert the

hydrophobic region (Nguyen et 01.. 1988). Although these processes are beginning to be

unraveled, there is still a lot to learn about them.

1.3d Protein Sorting between Membranes

A signal-anchor sequence of the OM (Tom70) has the potential to be recognized by

the IM and inserted in the absence of an OM (Li and Shore, 1992b). How are the OM and

LM able to distinguish proteins for insertion, that is, how are hydrophobic regions destined

to be stop-transfer or translocation arrest signals in the IM prevented from inserting into the

OM of the mitochondria'? The protein NADH-cytochrome bg reductase (NCBR) is located

in both the OM and the M S . About one-third of the molecules of NCBR are inserted into

the OM while the rest continue on to the IM where they arrest and are cleaved on the LMS

side and are released into the IMS as soluble proteins. Therefore, the protein has two

isozymes with different NH2-termini, in two different locations within the mitochondrion

(Hahne et a/., 1994). NCBR was defined to contain a "leaky stop-transfertt sequence. The

NHz-terminal sequence resembles that of Tom70 with a hydrophilic region of twelve

amino acids followed by a hydrophobic region of 39 amino acids. The targeting sequences

along with the components of the translocation machinery involved in this sorting

phenomena were investigated (Haucke et al., 1997). A lack of Tom7 caused more NCBR

to target to the OM, therefore, Tom7 is needed to allow the IMS sorted molecules to

continue to their destination. As well, Tim1 1 is needed to recognize the hydrophobic

region and target NCBR to the M S . Mutating two amino acids within the hydrophobic

region to weaken the hydrophobic character prevented insertion into the OM and increased

sorting to the IMS. Therefore, the hydrophobicity must also play a role in insertion of

potential transmembrane segments into the OM. As well, translocation arrest was

incomplete, because a small fraction of mutants were partially translocated to the matrix. I

would expect that this is due to the decreased hydrophobicity of the mutated signal, as seen

in Chapter 3, where potential transmembrane domains with a lower hydrophobicity are

translocated through both membranes into the matrix.

1.4 Phosphorylation and Mitochondria

One can now visualize the complexity of protein import into mitochondria. the signals

within the precursor proteins themselves, interacting with receptors within two different

membranes and with chaperones within two different cellular compartments. The cell and

the mitochondrion have an elaborate system to ensure the correct targeting and location of

mitochondrial proteins which are encoded in the nucleus. But what other signals exist

between the nucleus and the mitochondrion? How do they communicate back and forth'?

Since the mitochondrion plays a role in so many cellular functions, it must be able to signal

the nucleus when more or less preproteins are needed to up- or down-regulate various

metabolic processes, such as lipid and heme synthesis. mitochondria1 transcription and

translation, electron transport and oxidative phosphorylation. Most of the proteins

involved in these processes are encoded within the nucleus and in some instances, such as

respiration, the proteins involved are encoded within two genomes, the mitochondria1 and

the nuclear. As well, the transcription and translation of the mitochondrid encoded

proteins is regulated by nuclear encoded proteins and nuclear encoded assembly products

are needed to assemble some of the mitochondrial encoded proteins. The details of

communication from the mitochondrion to the nucleus are not clear, but seem to involve

metabolic signals or some son of signal transduction pathway which can reach within the

mitochondrion (Poyton and McEwen, 1996).

The regulation of cellular metabolism involves communication between and within

cells which is often achieved through signal transduction pathways (For reviews see

Cohen, 1992; Hunter, 1995). These signals can be transduced through enzymatic

phosphorylation and dephosphorylation of proteins in a sequential cascade (Krebs, 1994)

which reversibly alters their structural and functional properties. Phosphorylation and

dephosphorylation of proteins is achieved by protein kinases and phosphatases,

respectively. Phosphate groups are added by kinases to specific amino acids, most

commonly, serine, tyrosine and threonine, which then induce changes in protein activity,

localization or binding properties. Conversely, protein phosphatases remove the phosphate

groups. thereby regulating the duration and strength of the phosphorylation which

determines the physiological response. It has been estimated that humans may have as

many as 2000 protein kinase genes and up to 1000 protein phosphatase genes (Hunter,

1995). Therefore, it is reasonable to suspect that communication between the nucleus and

the mitochondria may involve one or more protein kinase, which along with some of their

target proteins may reside within mitochondria.

1.4a Kinases and Phosphoproteins in Mitochondria

Indeed, it has been shown that the regulation of mitochondria1 branched-chain a-

ketoacid dehydrogenase (Paxton and Harris, 1982) and pyruvate dehydrogenase (Linn et

ul., 1969) involves phosphorylation. Branched-chain a-ketoacid dehydrogenase complex

and pyruvate dehydrogenase complex play a role in the catabolism of valine, leucine and

isoleucine (branched chain amino acids) and in the oxidation of pyruvate, respectively.

Both are multicornponent complexes located within the mitochondria1 matrix. They are

each regulated by specific kinases, which inactivate them by phosphorylation on the

dehydrogenase component, and phosphatases, which along with the kinases, are closely

associated with each complex. Their distinct kinases which are encoded in the nucleus,

have been cloned from rat heart and they contain sequence similarity to members of the

prokaryotic histidine kinase family (Popov et a!., 1992; 1993).

The presence of other kinases within mitochondria has also been reponed. These

include CAMP-dependent protein kinases which have been shown to be located within

mitochondria by several groups (Henriksson and lergil, 1979; Dimino er of., 198 1;

Burgess and Yamada, 1987; Schwoch et al.. 1990; Vallejo et al. , 1997). Most of these

studies used purified. fractionated mitochondria to localize the kinase activity. although one

group used electron microscopy techniques (Schwoch et al., 1990). The general consensus

is that the CAMP-dependent protein kinase activity is located in the matrix or the matrix/LM

boundary. Although all fractions of the mitochondria have been reponed to contain the

activity.

CAMP-independent protein kinases have been reported to be present in mitochondria

as well (Clari et izl., 1976; Vardanis, 1977; Kitagawa and Racker, 1982). These groups

used purified, fractionated mitochondria followed by chromatography to determine that

there are two different CAMP-independent protein kinases present within the mitochondria1

membranes. The kinase purified by Vardanis is located in the IM. Tyrosine kinase activity

has been reported to be associated with the OM of mitochondria along with serine.

threonine kinase activities which were present in the Wmatrix fraction (Piedemonte rt al.,

1986; 1988). Casein kinase II activity has been reponed in rnitochondrial extracts ix. the

soluble fraction (Damuni and Reed, 1988) as well as in the membranes (Vallejo et al.,

1997). As well, protamine kinase activity has been shown in the soluble fraction of

mitochondria (Darnuni and Reed, 1988) and phosphorylase kinase activity has been shown

in the IMS and the membranes of brain mitochondria (Psam and Sotiroudis, 1996).

Therefore, many different kinases have been reponed to be present in several

compartments of the mitochondria. It remains to be determined whether or not all of these

reponed kinases are unique or whether there is overlap in their identities. As well their

target proteins and the role which they play in mitochondria1 function has not yet been

elucidated.

In addition to examining the kinases present in mitochondria, many different groups

have looked at phosphorylated proteins within the mitochondrion (Henriksson and Jergil,

1979; Backer et 01.. 1986; Muller and Bandlow, 1987; Piedemonte et al., 1988; Ferrari et

al., 1990; Sommarin er al., 1990; Pical et al., 1993; Rindress et al., 1993; Technicova-

Dobrova et 01.. 1993: H&ansson and Allen. 1995: Rahman and Hudson, 1995). These

studies have been carried out with different sources of mitochondria, mammals, yeast and

plants, with different fractions of the mitochondria and with different additions to the

reactions. Therefore the numbers and molecular weights of the resulting phosphoproteins

reported are quite varied to say the least. To date, however, only three proteins have been

routinely identified as being phosphoproteins: the a-subunit of pyruvate dehydrogenase

(Linn rt 01.. 1969), the a -subunit of the branched-chain a-ketoacid dehydrogenase

(Paxton and Hmis, 1982) and the autophosphorylated subunit of succinyl-CoA synthetase

(Steiner and Smith, 198 1). Of the other phosphoproteins reported, only three of these have

been identified, a 20 kDa fragment corresponding to cytokeratin type 11 (Gorlach e t ul.,

1995). an 18 kDa Complex 1 protein, (IP) AQDQ subunit (Papa et of., 1996) and

cytochrome c oxidase subunit IV (Steenaart and Shore, 1997b; See Chapter 4). All three of

these molecules are located in the IM.

1.5 Rationale and Objectives

The objectives of this work were to analyze topogenic signals involved in the

targeting of preproteins to the mitochondria and to identify novel mitochondria1 membrane

proteins that are potential targets for phosphorylation. Specifically, the features of signal-

anchor sequences that are involved in the retention of the signal-anchor and thereby

influencing the orientation of the signal-anchor in the OM of the mitochondrion were

investigated. As well, how apparently similar stretches of hydrophobic amino acids within

precursor proteins can function to target proteins to different locations within the

mitochondrion were determined.

The Tom70 signal-anchor sequence was shown to contain the targeting information

needed to insert it into the OM of mitochondria with an Nin orientation. Combining this

signal-anchor with a MTS or combining a heterologous stop-transfer sequence with a MTS

caused the proteins to insert into the OM with the opposite orientation (Gin). Therefore, it

was of interest to determine which feature(s1 of these signal-anchors caused the reversal of

orientation of the proteins.

The same heterologous stop-transfer sequence when place downstream of the MTS

would cause the protein to insert into the IM with an Nin orientation. I wished :o

investigate whether a signal-anchor transmembrane region when placed in this context

would function in the same manner, and if not, what is the deciding feature to cause a

hydrophobic region to insert into the IM. Answering some of these questions would help

to elucidate the signals involved in the sorting of mitochondrial proteins between

membranes and also, which features would cause certain orientations in the membranes.

Also, it was of interest to identify potentially phosphory lated mitochondrial

membrane proteins since they could be involved in signal rransduction pathways.

Communication between the nucleus and the mitochondrion and vice versa could involve

signal transduction pathways. Although a number of mitochondrial proteins have been

shown to be phosphorylated, to date very few of them have been identified.

Note: The references for the Introduction, the Discussion and the Appendix are found at

the end of the Discussion section.

CHAPTER 2:

An Amphiphilic Lipid-Binding Domain Influences the Topology of a

Signal-Anchor Sequence in the Mitochondria1 Outer Membrane.

An Amphiphilic Lipid-Binding Domain Influences the Topology of a Signal-Anchor Sequence in the Mitochondria1 Outer Membrane'

Nancy A. E. Steenaart.' John R. Silvius. and Gordon C. Shore*

.A~STRACT: blas7Op is tar~ered and inserted into the mi tachonririal outer membrane in the N,,-C,! ,,, orientation. via an NH2-terminal signal-anchor sequence. The signal-anchor is comprised of two domains: an NH:-terminal hydrophilic region which is positively charged (amino acids 1 - 10). followed by the predicted trdnsmembnlne segment (amino acids 1 1 -29). Substitution of the NH:-terminal hydrophilic domain with a matrix-targeting signal caused the signal-anchor to adopt the reverse orientation in the membnne tNcbto-Cln). This subs~itution resuIted in an increase in the net positive charge of the hydrophilic region. from +4 to +X. In contr:ist to the endaplasmic reticulum and the bacterial inner membrane. where the net positive charge is an important determinant in conferring protein topology in the lipid bilayer. we show hen that the reversal o f the Mas7Op signal-anchor was not due to differences in the number and positions of basic amino acids in the hydrophilic domain. However. a reduction in the hydrophobic moment of predicted amphiphi I ic helices containing an arginine. obtained by convert ins the apolar amino acids flankins the arginine to polar residues. caused the otherwise N,,(,,-C,, signal-anchor to re-adopt the original N,,-C,,,,, orientation uf Mas70p. The reduced hydrophobic moment at the NH2- terminus significantly reduced the ability of this domain to bind to synthetic liposomes whost. lipid composition retlectcd that of the outer membrane. These results identify amphiphilicity as an important determinant in causing retention of the NH1-terminus of a mitochondri;d signal-anchor on the cytosolic side of the outer membrane. tn addition to potential interactions between this domain and cytosolic- exposed cimponents of the import machinery. this retention may result as well ti-cmt interaction of the XH:-terminus with the surroundins membrane surtice.

The topology of integral membrane proteins is largely detcrniined during thcir biogenesis I Bloht.1. I9HO: Singer. IWOr. [ n panicular. the lkst domain that is inserted into the lipid bilayr is of parmount importance. since the orientation o f this segment will predetermine the orientatinn trt' all subsequent insertion events. In cases where the transmembrane segment is hydrophobic and contimns to a predicted a-helix. it can be classit?ed into two types. ;iccording to its function during polypeptide chain translo- c;ltic)n r Blobel. IC)XO: W ickner cYr Lodish. 1985: Singer. 10901: .srtprnrrisf~r .srqrrrnce.s. which do not carry intrinsic membrane-selecti ve targeting information. but rdther abrogate polypeptide translocation across the membrane that has been initiated by a proxima1 signal sequence: and si,qnd-otdwr .seqlrrncr.s. in which the targeting and mem brine-anchor ( wp-transfer) functions arc combined into one sequence. Signal-anchor sequences also carry inhrrnation that deter- mines their orientation in the membrme.

For the endoplasrnic reticulum t ER).' the determinants within the signal-anchor sequence that specify transbilayer orientation have been extensively investigated. by both

' This study was financed by t~pemting gmnts from the IMcJical Research Council and National Cancer Institute of Canada.

a Comsponding author. Telephone: t 5 14, 398-732. Fax: r 5 141 il)H-73x4.

Recipient o f a studentship fmm the Funds pour la Formtrtion de Chrrcheur crt I'Aidr ci Ia Recherche rFC.4R). '' Abstract published in .-\tlirrncr .4CS Abstrcwr.s. b l m h 1 . 19%. ' Abbrcviiltions: ER. endoplasmic reticulum: CCCP. carbonyl

cyanide m-chlorophenylhyclrc~.one: DHFR. Jihydrofdate rcductase: pOCT. pre-ornithine c;lrbamyl tnnsferue.

t..uperirnental rind statistical analyses. And although cxcep- tions do exist (.4ndrcws tlt 11.. 1992). i t is likely that net charge flanking the hydrophobic core of the signal anchor is o f primary importance rvon Heijne cYr Grrvcl. 1988: Hanmann et al.. I C > W Beltzer ttt 11.. 1Wl 1. especially as this reliites to the number and distribution of positively- charged residues on the NH:-terminal side (Park3 & Lamb. I99 I , lW3 ). Li kcwise. positively-c.har2t‘t.l amino acids art. an imponant topological determinant tbr integral mem br;m proteins of the bacterial inner membrane ( bun Heijnc.. 1986: Boyd & Beckwith. 10C)O). with the distribution of thest' positively-charged residues within the polypeptide cvnfmm- ing to the "positive insidr (cytosolic) rule" (von Heijne. (986). lndccd. mutations that violate this rule in polytopic constructs can be so disruptive that insertion of an otherwise transmembrane segment is bypassed in order to reestablish the positive-inside constraint (Gafvelin &k von Heijne. 1994). For both the ER and bacterial inner membrane. these determinants of tmnsbilayer orientilriun may function as retention signals cvon Heijne. 1986; von Hrlijne & Gavel. 1988; Hanmrtnn et al.. 1939: Boyd S: Beckwith. 1900: Btlltzer et 31.. 199 I : Parks & Lamb. 199 I . 1993: Gafvetin & von Heijne. 1994). In bacteria. this is due in put to the eIectrochemica1 potential across the inner membr~ne t insidr negative) (Andersson & von Heijne. 1994). and for the ER perhaps due to a potential and/or a specitic binding site for the retention determinants on the cis side of the membnne (Audicgier et a1.. 1987: ffartmann et 31.. 1989: Parks & Lamb. 1993 1.

19% American Chemical Society

In contrast to the ER and bacterial inner membrane, the available information on assembly of integral proteins into mitochondrial membranes is rudimentary. However, the principles that have emerged for the ER and bacterial inner membrane are likely to apply here as well (Blobel, 1980: Singer. 1990: Singer & Yaffe. 1990: Shore et al.. 1995). including the existence of domains that have been character- ized as stop-transfer (Hae et al., 1984: Nguyen & Shore, 1987: Nguyrn et al.. 1988: Glaser et al., 1990; Miller & Cumsky. 1993) and signal-anchor (Li & Shore. 1992a; McBride et a1.. 1992) sequences. Details of the latter have emerged from studies of yeast Mas70p. a bitopic protein of the mitochondrial outer membrane that adopts an Nin-Ccyto orientation (Hase zt al.. 1983). leaving the bulk of the polypptide Facing the cytosoi where it functions as a protein import receptor (Hines et al.. 1990). Characteristics of the Mas70p topogenic domain are strikingly analogous, both \tn~ctumlly and funcrionally. to ER signal-anchor sequences ( t i & Shore, I992a: McBride et al., 1992: Shore et al., 1995). ~tnd for this reason the nomenclature was retained (McBride et 11.. 19921. In the context of mitochondria, a signal-anchor wquence guarantees selection of the outer membrane during impon because it is predicted to trigger the release of the translocating polypeptide into the surrounding lipid bilayer prior to any possibility for commitment of translocation into the interior of the organelle (McBride et at., 1992). Ad- ditionally. the blas70p signal-anchor controls the orientation of insertion t Li & Sliore, 1992a) and contributes to the formation of protein oligomers (Millar & Shore. 1993. 1994).

The Mas7Op signal-anchor sequence contains a positively- charged hydrophilic domain (amino acids I - 10) followed by the predicted transmembrane segment (amino acids 1 1 - 29 I t Hase et a]., 198-l). The transmembrane segment is reqt~ird for targeting and insertion, whereas the hydrophilic domain cooperates with the transmembrane segment to increase thc. nte of import (McBride et al., 1992). Substitu- tion of the hydrophilic NH2-terminus with one containing the matrix-targeting signal of pre-ornithine carbamyl trans- ferast: (pOCT) resulted in the signal-anchor adopting the reverse orientation in the outer membrane (i.e., N,,,,-C,,) ( t i cYr Shore. 1992a). Similarly. introduction of a heterologous wp-trmsfer sequence immediately downstream of the matrix-targeting signal in pOCT created the functional equivalent of a signal-anchor sequence. and caused this otherwise matrix-destined protein to insen into the outer membrane, again in the Ncylu-C,n orientation (Nguyen et al., 1988). Together. these results imply that the matrix-targeting signal contributed a retention function to the NH2-terminus of the signal-anchor during protein translocation.

In the present study, we have analyzed the molecular determinants that specify this retention function and dem- onstrate that i t is the zlmphiphilic character of this retention sequence, rather than the number and positions of positively- charged amino acid residues, which results in the Nqlo-C,n orientation of h e signal-anchor. The amphiphilic positively- charged sequence has Iipid-binding chmcteristics, implying that the membrane surface could contribute in the retention of the N-terminus on the cytosolic side of the membrane.

MATERIALS AND METHODS

General Procedrrres. Previous articles describe the routine procedures used in this study [Li and Shore (1992a), McBride

et d. ( 1992). and Millar and Shore ( 1993) and references cited therein). These include in rirro transcription of pSPW plasmids. translation of the resulting mRNA in nbbit reticulocyte lysate in the presence of jJ5S Imethionine. purification of mitochondria from rit heart. protein import in vitro, and analysis of import products by SDS -P.GE and fluorography .

Mitochondria1 lrnporr. Reaction mixtures contained 10% (v/v) rabbit reticulocyte ly sate transcription - translation products fabeled with [%]methionine. mitochondria (0.5 rng of proteidml), 0.125 rnM sucrose. 32 mM KC]. 0 3 n M magnesium acetate. 9.0 mM Hepes. pH 7.5. 0.5 mM dithiothreitol, 0.5 mM ATP. 2.5 rnM sodium succinate. 0.04 mM ADP. and 1.0 mM potassium phosphate. pH 7.5 I MRM/ KMH). Some reaction mixtures also contained I p b l carbonyl cyanide m-chloropheny Ihydrazone t CCCP) as in- dicated in the figure legends. After incubation at 30 'C for 30 rnin, the mitochondria were collctted by centrifugation for 3 min at 12000~ and resuspended to 0.5 mg uf protein/ mL in MRM/KMH f 1 !tM CCCP. For post-trypsin treatment. these mitochondria were incubated with tqpsin (0.125 mg/mL) for 20 min on ice after which soybean trypsin inhibitor (1.25 mg/mLj was added and the incubation continued for 10 rnin. Mitochondria were recovered by layering 50 pL aliquots over a 750 p L sucrose cushion (0.25 mM sucrose. 10 mM Hepes. pH 7.5. and 1.0 mM dithio- threitol) and centrifuging at 12000g for 6 min. Pellets were prepared for SDS -PAGE either directly or after extrxtinp in alkali. For the latter. the mitochondria were resuspended in freshly prepared 0.1 M Na2CO~, pH 1 1.5. to finat concentration of 0.25 mg/mL and incubated on ice for 30 min with periodic vonexing. Membmnes were co1lectt.d by centrifugation at 30 psi for I0 min in a Beckman clirfuge (Beckman Instruments. Carlsbad, CA 1.

Plusmids. The plasmids. pSP(pOMDZ9) tLi & Shorc. 199%) and pSP(p0-OMD) tLi cYr Shore. 19923). were manipulated by standard PCR techniques to create the 13 other constructs employed in this study. The correspondins amino acid changes to the various topogenic domains are described in the figure lezends. The authenticity uf all DNA constructs was verified by nucleotide sequencing.

RESULTS AND DISCUSSION

The hybrid proteins. pOMD29 and PO-OMD, are de- scribed in Figure I . pOMD29 (Li & Shore. I9Xb 3 contains the signal-anchor sequence of yeast Mas70p (amino acids 1-29) fused through a glycine to amino acids J- 156 of dihydrofolate reductase (DHFR). pO-OMD (Li & Shore. 1992a). on the other hand. was created by replacing amino acids 1-1 l of the Mas7Op signal-anchor sequence in pOMD29 with amino acids 1-38 of rat liver pOCT. a domain which contains a potent matrix-tar~eting signal (Nguyen et al.. 1986). Targeting and insertion of the two hybrid proteins into the outer membrane of intact mitochon- dria in uitro have been previously characterized ( Li & Shore. 1992a.b: McBride et al.. 1992: MilIar & Shore. 1993. 1994). [n the case of pOMD29. the bulk of the protein was accessible to external protease following import. indicating an Nin-C,,,, orientation (Li & Shore. 1992a.b: McBride et al., 1992) (Figure 1B). In contrast. pO-OMD adopted the reverse orientation in the outer membrane (i-r., N,,,,-C,,, Figure 1B) (Li & Shore, 1992a). Treatment with externai

c n o

OMM

IMS

FIC~L'RE I : pOklD19 and PO-OMD. ( r\J p0-0MD was derived tiom pOMD211 by replacing amino acid% I - I I of pOklDZ9 t i.r., c.urrc~pondin~ to amino ;icd> I - 1 I 01' blas7Op; Hasc ct d.. I9X-lI ~vith amino acids 1-38 of pOCT tNguycn e[ a].. 1YHh). The predicted trdnmcmbranc q m e n t i s in boldfxe type and under- lined. .-Irrow. [he rite in [hc pOCT 4gnal requcncc where protcolytic proccshing HOUI~ othcnvise fake p l x t in the matrix; DHFR. amino acids S- 1 M of mouw dihydroti)l;ttc rcductasc I \ce .Llateri;ils and Jlcthuds): pluws. pwitivcly-charged amino acidh; numbers, amino acid pohitions relat i~e to the initiator rncthioninc, I B I Topology of pOMDI9 and p0-0h lD in the mitochondria1 outer membr~nc ( L i & Shim. 1W2x .CIcBride et a].. IWZ). CYTO. cytosol; OMM. wtcr n~itochondrial nicmbmnc: IMS. ~nrcrmembmnc spacc. See text ti)r funher cictailh.

protease fdlowing import o f PO-OMD resulted in clipping and remo\,al of an -2 kDa fragment from the protein. The frqnient corresponded t i ) the NH2-tcrminal pOCT matrix-

@ targeting signal (Li & Short.. I992a). Upon disruption ot' the outer membrane by usmutic shock. the external protease gained access tt1 the intermembrane space and degraded the remainder of the p0-0MD polypeptide. (Li 8 Shore, l1W2a).

Impon of pOklD20 and PO-WID in titro was ATP- dcpendtrnt and temperature-.;c.nsitive t Li LYr Shore. IY92a.b; McBride et 31.. I99Z). %lorcover. similar to all outer membrane proteins analyzed to date and in contrast to most inner membrane and matrix proreins Attmli & Schafz. 1988: H a d et 11.. I Y X O ) , insertion o fpOMDZ9 and PO-OMD into the mitochondrial outer membr~ne occurred in the presence of the uncoupler. CCCP and. therefore. did not require cooperation of the e!ectrochemical potential across the inner membranc during impon (Li & Shore, 19L)Za.b). [n the absence of CCCP. however. a small tixtion of the PO-OMD that was associated with the mitochondria had the pOCT 5ignal sequence removed by the matrix processing machin- ery. indicating that at least the NH2-terminus of these molecules had reached the matrix. This may result from the mitochondria1 prepttrrrtion containing a small percentage of organelles with a ruptured outer membrane. Exposed regions of the inner membrane in such damaged mitochon- dria have the ability to directly import pOCT at an efficiency which is in hrct greater than that of intact mitochondria (Li & Shore. l992b: unpublishedl. It is also possible that the potent matrix-targeting signal can partially override the signal-anchor and deliver a fraction of the molecules to the interior of the orpnelle ~Hahne et al.. 1993). To avoid this problem. a11 measurements of impon into the outer mem- bnne in both this md the previous study (Li & Shore. 1992) were performed in the presence of CCCP. which abolishes this alternative pathway.

F ~ R E 2: Schcmrrtic illustntion of structur~l altcrationh to the NHI- trrminal hydrophilic domain o i the signal-anchor sequence o i pOMD2Y and PO-OMD. In series :I. the f o l l o~ ing amino acid icquenccs were introduced between rcsidues I 0 and I I of pOMD24): glycine-threonint: cpOhlDZ9 kr; amino acids 2 - 10 o f pOMD2Y tlanked on srther stde by glyc~ne-thrcontnr c pUMD29 R); a iequence specitled by codons for amino acids 2 - I 0 o f pOMDZ!I read in the reverse direction ( CLVPCNEALI and tlltnkrd on either side by glycinr-thrconine 1pOhlD29 R'I. In irries B. site- directed mutagenrsis ofp0-0MD was pcrfirmed to crmwrt lysines at positions 1 I and 16 to ~spxrrgines and at position 24) to glutarninr (PO-OMD K3). Additionally. the urginine at position 26 111 PO- OMD K3 was convened to ~lulclmine (PO-OMD K R J I . 31ack recungle. predicted trmsmembnnr rqment of the pOMD39 signal- anchor sequence (amino acids 1 1 -24)): pluses and minuws. positivrly and negatively charged rosidutrs. rcspcctivcly: numbers. m i n o acid positions relative ro thc initiator methioninc t i ~ r squence~ that derivc from Mas7Op (pObIDZ9 and pO-OMD) and pQCT ( PO-OIbID): ORIESTATION IN Oh.1h.I. hummarir.cs result5 of Figure 3 in which the qignal-anchor squt'nce inserted into [he outer mitochondria1 memhranr (0hIh.l J in either the N,l,-Cc,l,, t N,,) or the N,!a,-C,n ( N,w,) orirntation; IMPORT OF AThIS. summarizes the wsults of Figure -I. in wh~ch the trmsrncmbrrrnr wsrncnt tTXIS I was deleted ( A I from each construct. and the htt. of the polypeptide ti)llowing impan into intact mitochondria was Jetcrmined. Matrix. import to the matrix; None. no obscnahlt. impon [we Figurt. 4).

In cimverting pOMD29 to pa-OMD. both structural and functional alterdtions were made to the NH2-terminal hy- drophilic domain of the blas70p signal-anchor sequence t Figure I ). These included an increase in length (from I0 to 37 amino acids). an increase in net charge t from +-I to 4-81. and a change from a domain that by itself exhibits negligible. or weak. targeting information t Mas70p amino acids 1 - 10 in pOMDZ9) with whole intact mitochondria (Hun et 31.. 1984; Li & Shore. 1992b: McBride et al.. l 9YZ to one that contains a potent matrix-targeting signal (pOCT amino acids 1-32 in PO-OMD) (Nguyen et al.. 1986).

To investigate the contribution of net charge o n the orientation of the protein in the mitochondria1 outer mem- brmr following import. a series of changes were made to the hydrophilic NHz-terminus of pOblD3,Y that lengthened this domain and increased the number of positive1 y-charged residues. This was achieved by duplicating the codons that specify the hydrophilic domain. either as a direct DNA repeat (pOMD29 R. in which the net charge of the encoded hydrophilic NH2-terminal domain was increased from +4 to +7. Figure 2A) or as an inverted DNA repeat (pOMD29 R'. in which the number of encoded positively-charged residues was left unchanged. and a single negatively-charged residue was introduced into the second repeat. Figure 'A). Conversely. site-directed mutapes i s was performed on the NH2-terminal hydrophilic domain of PO-OMD to convert the three lysine residues at positions I 1. 16. and 29 to either

Mitos

Alkali

Post-Trypsin

CCCP

- + + Mitos + + + Alkali + + + CCCP

FIL;L+RE 3: Insertion of pOMDZ9 m d PO-OMD into the mitochondria1 outer membrane. ( .A) Standard import rextionh c.mraincd ["SI- pOMD29 (upper) or ['%lpO-OhtD !lower) and were carried out at J "C (lane 3) or 30 'C (lanes 2 and 4) in [lie p r c w ~ c e (lant.!, 3 and 4 1 or absence of mitochondria r Mitos~ (lane 2 ) and in the presence of 1.0 y M CCCP I lanes 2-4 I . Fdluw mg recuvery b~ crntri t'ugmon. pellets were subjected to extrxtion with 0.1 M Nrr2CO~. pH 11.5 (Alkali) (lanes 2-41, and the insduble rnarrrtal was ~ n a l ~ ~ e d by SDS- PAGE and tluorography. Lane I . 10% of input rdioactive precursor protein. i B) As in ( . - \ I except that the indicrttrd [ "Sjprecursor protcins (described in Figure 2) were examined. After impon, mitochondria were either extr~cted with alkali (lane 31 t)r hubjrcrttd to proteolysis with trypsin (0.125 mg/mL) (Post-Trypsin) (lane 4). The asterisk denotes trypsin-clipped prtrcursc)r protein.

asparaghe or elutamine (PO-OMD K3), and. additionally. to convert the arginine residue at position 26 to glutamine (PO-OMD KRJ) (Figure 26). In this way, the net charge of the NW2-terminal hydrophilic domain was reduced from +8 (PO-OMD) to either +5 (PO-OMD K3) or +4 (PO-OMD KR-S).

Orirnturion oj' Muranr Proteins irl the Mirochondrial Oirrvr ,Mmhrune. The various protein constructs are described schematically in Figure 2 and their import into the mito- chondrial outer membrane examined in Figure 3. Impon in lrirro was assayed by the acquisition of resistance to extraction at alkaline pH which. for both pOMD29 and pO- OMD. was dependent on the presence of mitochondria in the import reaction (Figure 3A. compare lanes 2 and I), and was temperature-sensitive (Figure 3A. compare lanes 3 and 4). Orientation of the membrane-inserted polypeptides was determined based on their accessibility to exogenous trypsin,

as previously documented tLi k Shore. I 992a~ For pOMD29. proteolysis resulted in degradation of the polypep- tide (Figure 3B. top panel. lane 4). consistent with its N,,- C,,,,, topology (Figure 10: Li & Shore, 1992a.b; McBride et al.. 19921. In contrast. trypsin treatment of imported pO- OMD resulted in the removal of an -2 kDa frqment from the NHz-terminus of the protein (Figure 3B. lower panel. lane 3). due to the N,,,,,-C,, orientation of the protein (Figure 1B: Li & Shore. 1992a1. In Figure 3B. the dipped form is denoted by an asterisk. In the absence of mitochondria. pO- OMD was completely degrrrded by trypsin [not shown: see also Li and Shore ( I992a)j. In both this (Figure 3) and the previous study (Li & Shore. 1992s). however. clipping of imported PO-OMD was incomplete. and a population of full- length PO-OMD was observed. It is possible that this constitutes PO-OMD in the Pi,,,,& orienration. but where the NHrterrninus is shielded from protnse.

Mitos

Post-Trypstn

CCCP

The pttern of import c)f the mutant pOM D Y pol) peptides I I .c.. pOMD20 k. pOhlD29 R. ml p O h l D 3 R'I was hiniilar lo that 01' iviId-t!ye pOh.lDZC) t Figurc 3B. upper pi1nc.I I. ,-\cqui\i~ion of resistance 111 txtr;~ction b! alkali ii.35 depenci- c'nt 1111 1111' prcsencc of mitoch0nC1ri;t ( Figurc 3B. lancts 2 m d 3 ,. and following rnt.nihr;ine insertion. the bulk of the prdypepticle~ ucre ;t~'ccssihle to I ' Y C ) ~ C ~ O L I ~ try p i n ( Figurc .?B. I:lne 4 I. indic;lting ;in N,,,-C,,,,, orient;ition. lncreaing the net chargc of the hytlrophilic NH:-ternminus of the bti1s70p 4gnal-anchor sequence from +-I ( pOM DZ1l ti, +7 r pOlLlDZ9 RI. therefore. h;~d no effect un the urientatir~n of the protein in the cwter mernhrane.

Likewise. decreasing rht. net chrlrge w~thin the NH:- remiinus o f p0-0MD f'rum t~ tu +5 (p0-0klD K.3 r or to -4 tp0-0MD KR4) did not alter import of the prottin. or the nature of its i~ccessibility to exogenuus trypsin t F i p r c 3B. loww p;tnel): i . tp . . the protein retained the N,,,,,-C,, t~rientation in the outer msmbr;tne.

Dth~;o11 o f t h r ~ - t i ~ ~ . s r t r t ~ r t r f ~ r ~ ~ ~ ~ ~ ~ Dormiitr tj-orj~ tlrc pOMD2Y (ICIJ PO-OMD Corrsrrur-rs. To exitmint. the crffects of the vxious charge mutiltions on the independent ability of the h~drophilic XH2-terminal domains of pOMD29 and PO- OMD to import the protein to the mitochondria1 matrix. the 19 amino acid transmembrane domain was deleted tiom d l constructs (see Figure 2).

[t had previously been estabiished that amino acids 1 - 15 of the Mas70p signal-mchor. when fused directly to DHFR. exhibited negligible activity as a matrix import signal (McBride st al.. 1992). and this was also true using a mature matrix protein (OCT) as the passenger molecule. in place of DHFR (unpublished). Likewise. removal of the transmem- bnne domain from pOMD29 k. pOMD29 R. and pOMD29 R' resulted in proteins (each designated by a A symbol) that were capable of binding to the surface of mitochondria (Figure 4. upper panels. lane 3). but did not cross into the interior of the organelle (Figure 1. compare lanes 3 md 3):

i.e.. the!. rcrn;lind accessihlc to trypin. None ot' [he crmsrructs iverc resistant to c.utr;iction by ;llkali I not \ f iou n 1.

Incre;~sing thc nct ch;~rge of rhe NH:-tc.rniinaI hyclrophilic domain ut' the kla37Op zignal-;rnchor. therefore. did not ctmven this hrtiain to :I matrix-t;trgeting zlgnai.

Correspondingly. ;t rctltrctton in net ch;lr:t' of' thc NI-{:- tctm~inal domain c)f p0-OhlD Ii.tl.. the pOCT \ i y x i l w- qucnccr from +$ to f 4 did not prewnr its ability 11) t;lrgct DHFR to the matrix in the ahsencc uf ;I tr;~nsmembranr. domain t Figure 1, lower p;lnels). Follu\ving import. both wild-t)p ;rnd mutant proteins \rere prc)cessed ( Figure 4. lane 3). the processed products but not the t'ull-length prccurwr poly peptides uyre prc~tecttld from esogenouh t p p i n ( Figure 4. 1;tnc 1). and this protection was depentknt on thc elc.ctrochernicrtl potentid across the inner membrane ( Figure 4. lane 51. .-\I1 of these thiings arc consistent with import to the matrix. Compared to pO-OIMD A. however. the initial rates of import of the proteins to the matrix were ciecrcased by - 1.5- and -2.5-fold for p0-0MD K.3 A ;mi p0-0MD K R I A. respectively (not shoivn).

.-hrplriplrili~. Donr~titr Dcrc~rnritrr~.~ rlie .V, .,,,,-C,,, C)r-ic*~lr~~riotr (g' rlrr Si,qtt~rl-Arr~-itor. Taken together. the results for pOMD29 and PO-OMD and their w i o u s mutant derivatives indicate that retention of the YH2-terminus of the signal- anchor on the cytosolic side of the outer membrane during import correlates. unexpectedly. with the ability of this domain to function independently as a matrix-tarseting sequence. .A common property of matrix-targeting signals that might relate to a retention function. however. is their ability to adopt an amphiphilic helix. To examine this. the NH:-terminus of PO-OMD KR4 was manipulated to convert apolar residues tlanking the three arginines in this sequence (positions 6. 15. and 23) to polar amino acids (glutmine or serine, Figure 5A). While such changes are compatible with an a-helix (Chou & Fasman. 1974). they would be expected

OOL OB 09 OP 02 0

FK;L:KE 7: Oricntdtion 01- signal-anchor sequences in the mitochon- clrial outer mcrnb~mc. The import machincry is tfepictcd as ztipplcd \hapt.s in ihc outer rncrnbrunc. ~ i t h thc regions cp1)st.d to the cylnolic sidc rcprcscnting rhc import receptor comples 1 for rrvicws. scc Hannai.? ct at. I IW3) and Kichlcr rt at. r 1993h)l. The model predict5 that orientation o1'an NH2-terminal signaI-anchor wqucncc in the menibrant. ih Jctcrmincd by thr prcsrncc or abscncr of u rctcnrion ~ignrtl upstrcm of rhc rr~nsrncmbrxw st.;mctnt I black cy linrlcrs 1. (-4) A \ lc)u ratc of' dissociation I ~ITOU'S of the retention \ignal i'ron~ i t5 protein- and lipid-binding i res on thc I . I . ~ Grlc of the rncrnbrtlnc. rclatiic to the ratc of unfolding of the pc)lypeptidc on the other sidc ol'thc. transmcmbmnc sc~mcnt. rcsults in inscnion Jcrosh the bilaycr in rhc N,,,,,-C,,, uricnu1ii)n. (B) In the rrbwncc of an NHI-rcrniinal rcrcnrion stgnat. inhenion is in the t)ppositc N,,- C,,,,, oricntarion. See rcxt ti)r discus\ion. Coil. amphiphilic helix; C>IO. ~y1)zol: OMM. t ~ ~ t c r mirochondnal membrane: IX1S. intcr- mcn~branc \pilcc.

Honlingcr c't 31.. I W S ) within the impon receptor cump!ex Ifor rcviewh. see Hannrrvy ct 31. ( IW3) and Kiebler r't 11. ( ILlY3b 1 1 and with the surrounding membrrlne surhce. resulting in rctcntirm of this domain on the cytosolic side 01' the niernhrane durins import. b1orct)vt.r. interaction of this domain with the n~crnhrinc \ L I ~ ~ I L ' E would likely he mmi- festt'cf. as wclt. after import is completed. and might explain why about halfo1' the p0-0MD mc)lecules following import were shielded from trypsin (Figure 3). This is rcprt.st.ntt.d .;chen~atically in Figurc 7. Bccausr of the inter~ctions of the NH2-tetmini~l domain with protein- and lipid-binding sites o n the cis side of the membrane. import favors unfolding and delivery of the COOH-terminal region of the pmtein into thc translocation pore [for rcviews. see Kubrich et al. r IW5t and Lithgow et 31. ( 190511. resulting in insertion of the protein into the hi!;lyer in the N,,,,,-C,, orientation (Figure 7 . In the absence of mong interactions between the NH2- rerminus of the signal-anchor and the binding site on the cis side of the membrane. however. import Pavors the NH2- terminal domain entering the translocation pore. r~ the r than unfolding and translocation of the COOH-terminal region of the protein. and the protein adopts an N,,-C,,,, orientation t Figure 7B ). Furthermore, we have recently demonstrated that pOMD29 A (i.u.. containing the Mas7Op signaI-anchor lacking the transmembrane segment) binds to yeast mito- chondria bearing the MasZOp import receptor from either yeast (Ramage et al.. 1993) or human (Goping et al., 1995) sources, but does not bind to the lipid bilayer surface of mitochondria specificalIy deleted of this receptor (H. M. McBride. 1. S. Goping, and G. C. Shore, unpublished results). Despite the interaction of pOMD29 with h i s receptor. however, such binding is insufficient to retain the NH2- terminus on the cytosoIic side of the membrane following import (Figure 7%).

ACKNOWLEDGMENT

We are p t r f u l to Drs. I. S. Goping. R. MacKenzie. M. Nguyrn. J. OrIowski. and H. XIcBride and D. Millrtr for discussions and comments on the manuscript.

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CHAPTER 3:

Alteration of a Mitochondria1 Outer Membrane Signal Anchor Sequence

That Permits Its Insertion into the Inner Membrane. Contribution of

Hydrophobic Residues.

THE JOURNAL OF BIOLOG~CAL CHWWRY O 1997 by The h e r i a Society for Biochemistry and hfoleculru Biology. Inc

Vol. 272. No. 18, h u e of htay 2. pp. 12067-12061. 1997 Pnnfed in U S A

Alteration of a Mitochondria1 Outer Membrane Signal Anchor Sequence That Permits Its Insertion into the Inner Membrane CONTRIBUTION OF HYDROPHOBIC RESIDUES*

(Received for publication, December 20. 1996. and in revised form, February 8. 1997)

Nancy A. E. SteenaartS and Gordon C. Shore8 Fmm the Department of Biochemistry, hfcIntyre Medical Sciences Building, McGilf University, Montreal, Quebec H3G IY6, Canada

Tom'lOp is targeted and inserted into the mitochon- drial outer membrane in the N,,-C,,,, orientation, via an NH,-terminal signal anchor sequence. The signal anchor is comprised of two domains: an MI,-terminat hydro- philic region which is positively charged (amino acids 1-10) followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NltIy-terminal domain with a matrix-targeting signal caused the signal anchor to adopt the reverse orientation in the outer membrane (N,,,,-C,,) or, if presented to mitoplasts, t o arrest protein translocation at the inner membrane without insertion. Physically separating the transmem- brane segment from the matrix-targeting signal by mov- ing it downstream within the protein resulted in a fail- ure to arrest in either membrane, and consequently the protein was imported to the matrix. However, if the mean hydrophobicity of the Tom7Op transmembrane segment was increased in these constructs, the protein inserted into the inner membrane with an N,,-C,,, ori- entation. Therefore we have determined conditione that allow the Tom7Op transmembrane domain to insert in either membrane, pass through both membranes, or ar- rest without insertion in the inner membrane. These results identify the mean hydrophobicity of potential transmembrane domains within bitopic proteins as an important determinant for insertion into the mitochon- drial inner membrane.

Nuclear-encoded precursor proteins destined for import into mitochondria are sorted to one of four compartments in the organelle: outer or inner membrane, intermembrane space, or matrix. Since import of most of these proteins is mediated by a common protein translocation machinery (for reviews see Refs. 1 and 21, specificity for sorting must reside within topogenic domains present in the precursor protein. To date, four such domains have been characterized as follows: signal anchor sequences selective for protein insertion into the outer mem- brane ( 3 4 , stop-transfer sequences that arrest and integrate proteins in either the outer or inner membrane (6, 71, inter- membrane space-sorting signals (8, 91, and matrix-targeting signals (10, 11). In addition, complex variations of these do- mains may well exist, especially for proteins that assume poly- topic structures within the lipid biIayer of either the outer or

inner membrane, e.g. porin ( 12) and uncoupling protein (13, 141, respectively. With the exception of matrix-targeting sig- nals, which are characterized by sequences rich in basic and hydroxylated amino acids with the potential to form an am- phiphilic heIix (10, 15, 161, the others contain stretches of hydrophobic residues capable of spanning a membrane lipid bilayer.

Outer membrane signal anchor sequences combine the func- tion of targeting and membrane-anchoring into one sequence which also carries information that determines orientation. A well studied signal anchor sequence is that of Tom70p (3,4, 17), an outer membrane bitopic import receptor (18). The Tom70p signd anchor sequence contains a positively charged hydro- philic domain (amino acids 1-10) followed by the predicted transmembrane segment (amino acids 11-29) (19). The trans- membrane segment contains a11 the information needed to target and insert a fusion protein into the outer membrane with the same orientation as Tom7Op (Ni,-C,,,) (4). Signal anchor sequences control the orientation of insertion depend- ent on the hydrophilic NH, terminus i17,20) and can contrib- ute to the formation of protein oligomers (21,22). Signal anchor sequences can also function a t the COOH terminus of proteins such as Bcl-2 resulting in o Ci,-No,, orientation (23, 24).

Stop-transfer sequences do not contain intrinsic membrane- selective targeting information but rather they are passive transmembrane segments that are located downstream of mn- trk-targeting siga&. causing an otherwise matrix-destined protein to arrest translocation and insert into the outer or inner membrane. This is exemplified by yeast cytochrorne oxi- dase subunit Va which has been shown to reside in the inner membrane and whose sorting signals are consistent with an NH,-terminal matrix-targeting signal combined with a down- stream stop-transfer (7, 25). Introducing a heterologous stop- transfer sequence, derived from Vesicular stomatitis virus G protein (261, into pre-ornithine carbamoyltransferase (pOCT)' downstream of the matrix-targeting signal causes this other- wise matrix-destined protein to insert into the inner membrane (27). However, when it is placed contiguous to the matrix- targeting signal, the protein arrests in the outer membrane (28).

Likewise, intermembrane space-sorting signals contain a stretch of hydrophobic residues immediately downstream of a matrix-targeting signal (291, and indeed there is one example of a protein &th such a sequence, NADH-cytochrome-6, reduc- t&e, that sorts to both the outer membrane and intermem-

*This was "pwrted by manta the Resen* b r a e space (30). Controversy remains, however, concerning Council and the National Cancer Institute of Canada. The costs of publication of this b i d e were defrayed in part by the payment of page the pathway by proteins bearing intermembrane charges. This article must therefore be hereby marked 'advertisement" in ackrdnnce with 18 U.S.C. Section 1734 sdely to indicate this fact.

$ Recipient of a McGill FacuIty of Medicine Studentship. The abbreviations used are: pOCT, preornithine &amoyl trans- # To whom correspondence should be addressed. Tel.: 514-398-7282; ferase; CCCP, carbonyi cyanide m-chlomphenylhydrazone; DHFR, di-

Fax: 5 143987304; E-mail: sho~edcor.mcgill.ca. hydmfolate reductase.

his paper is available on line at hnp://wrmv-jbcsbnford.du/jbc/ 12057

12058 Relocating a Signal Anchor Sequence to the Inner Membrane

space-sorting signals. In one model the hydrophobic stretch is suggested to arrest translocation during unidirectional import within the inner membrane translocation machinery; process- ing of the precursor on either side of the membrane then liberates the mature protein into the intermembrane space (29). Another model proposes that following removal of the NH, terminus in the matrix, the hydrophobic domain redirects the precursor protein back from the matrix compartment to the intermembrane space (31). It has been consistently observed, however, that the hydrophobic domain, while capable of trans- location arrest within the inner membrane import machinery, does not integrate into the surrounding bilayer. Clearly, this domain is functionally different from the structurally analo- gous regions in signal anchor and stop-transfer sequences.

Here we have addressed the question of how apparently similar stretches of hydrophobic amino acids within precursor proteins can function to target proteins to different locations within the mitochondrion. To do so, we have determined con- ditions and modifications that result in the transmembrane segment of a signal anchor sequence being inserted into either the outer or inner membrane, passing across both membranes without being arrested, or being arrested across the inner membrane without inserting into the bilayer. This recapitula- tion of targeting of the hydrophobic domain to different com- partments was found to depend on several factors: 1) its net hydrophobicity, 2 ) whether or not it is permitted to pass across the outer membrane, 3 1 its distance from a matrix-targeting signal, and 4) the relative strength of the matrix-targeting signal.

M A T E R I U rlND METHODS General Procedures-Previous articles describe the routine proce-

dures uved in this study (Refs. 17 and 32 nnd the references cited therein). These include irr uirm trnnscription of pSP64 plasmids. trnns- lotion of the resulting mRNA in rnbbit reticulocyte lysate in the pres- ence of 1"%lmethionine, purification of mitochondria from rat heart and of mitoplasts from m t liver, protein import in oitro, nnd analysis of import products by SDS-polyacrylnmide gel electrophoresis nnd fluoroqnphy.

Mitochondrial Impon-Renction mixtures contained 1 0 8 (vh) rabbit reticulocyte lysate transcription-tmnslntion products lnbeled with ["Slmethionine, mitochondrin or mitoplosts (0.5 mg proteintml). 0.125 mar sucrose, 32 ma1 KCI, 0.8 mar rnngnesium acetate. 9.0 met Hepes, pH 7.5, 0.5 mar dithiothreitol, 0.5 mv ATP, 2.5 mat sodium succinate, 0.04 m~ ilDP, and 1.0 mr potassium phosphnte, pH 7.5. Some reaction mixtures also contained 1.0 gar cnrbonyl cyanide rn-chlorophenylhydra- zone (CCCP) ns indicated in the figure legends. The import reaction mixtures were incubated at 4 or 30 "C for 30 min. For past-trypain treatment, reaction mixtures were incubated with trypsin (0.125 mg/ mi) for 20 min on ice nRer which soybean trypsin inhibitor (1.25 mg/ml) wns ndded, nnd the incubation continued for 10 min. The mitochondria or mitoplasts were recovered by layering 50-4 aliquots over a 750-kl sucrose cushion (0.25 mlI sucrose, 10 mw Hepes, pH 7.5, 1.0 mnt dithi- othreitol) and centrifuging a t 12,000 x g for 6 min. Pellets were pre- pared for SDS-polyacrylamide gel electrophoresis either directly or after suspending in freshly prepnred 0.1 sf N%CO,, pH 11.5 (alkali), to a final concentration of 0.25 mg of proteinfml and incubating on ice for 30 rnin with periodic vortexing. Membranes were collected by centrifu- gation a t 30 p s i . for 10 min in n Beckman Airfuge (Beckman Instruments).

Plasmids-The plasmids, pSP(pOMD29) (3) and pSP(p0CT) (331, were manipuIated by standard polymerase chain reaction techniques to create pSRpOSA 1411, pSP(p0-SA 2421, pSP(p0-141A1, pSP(p0- 242A1, and pSP(p0-SA 14144). The plasmids pSP(p0-OMD1 (20) and pSP(p0-DHFR) (34) have been described previously. The corresponding polypeptides that are encoded by these plasmids are described in the figure legends. The authenticity of ail DNA constructs was verified by nudeo tide sequencing.

RESULTS AND DISCUSSION

3) PO-OHFR: N= DHFR 1 1 3w4 186

6) p b S A 242: NN DHFR 1 1 242 4 r 86

FIG. 1. Fusion protein constructs. Stnndnrd recombinant DNA nnd polymerase chain reaction methodologies were employed to con- struct pSP64 plasmids encoding various fusion proteins containing the following NH,-terminal domains fused to nmino acids 4-186 of mouse DHFR: 1) pOblD29 tLi and Shore 13)). amino acids 1-29 of yTom70p followed by Gly; 21 PO-SA 36 (formerly p0-0hID: Li and Shore (20) ) , amino ncids 1-36 of rat pOCT, Gly, nmino ncids 11-29 of flom7Op. nnd Gly-Pro; 3) PO-DHFR [Skerjonc et al. (3411, amino acids 1-36 of pOCT followed by Gly-Arg 4) PO-SA 141. nmino acids 1-141 of pOCT, amino acids 11-29 of flom70p. followed by Gly; 5 ) p0-141A is PO-SA 141 lacking nmino acids 11-29 of flom'i0p; 6 ) PO-SA 242, ns in number 4 except that the NH, terminus contains amino acids 1-242 of pOCT; 7) PO-242A is PO-SA 242 lacking nmino ncids 11-29 of yTom7Op. A1 convtructs were verified by nucleotide sequence analysis. Helix, matrix- tnrgeting signal of pOCT; shaded box, the signal anchor sequence of yTom70p cnmino ncids 11-29): boxed DHFR. amino ncids 4-186 of DHFR.

NH,-terminal signal anchor domain of Tom70p (amino acids 1-29) fused to dihydrofolate reductase (DHFR) and is targeted and inserted into the outer mitochondria1 membrane in the N,,-C,, orientation (Fig. 5 4 i3, 4, 20). Replacement of the extreme hydrophilic NH, terminus of the pOMD29 signal an- chor with the matrix-targeting signal of pOCT created PO-SA 36 (formerly PO-OMD), which inserts into the outer membrane in an orientation opposite that of pOMD29, i.e. N,,-C,, (Fig. 5A) (17, 20). Deletion of the predicted transmembrane portion (amino acids 11-29) of the pOMD29 signal anchor abolishes the ability of the protein to target mammalian mitochondria in uitm (41, whereas the PO-SA 36 fusion construct containing the pOCT matrix-targeting signal but lacking the Tom7Op trans- membrane segment (i.e. PO-DHFR) is efficiently imported to the matrix (34, 35) (Fig. 5A).

A Downstream Signal Anchor Transmembrane Segment Does Not Arrest Transport of a Matrixdestined Protein-In PO-SA 36, the transmembrane portion of the Tom70p signal anchor is contiguous to the pOCT matrix-targeting signal. To investigate the consequences of physically separating these domains, a spacer region was introduced by replacing pOCT amino acids 1-36 in PO-SA 36 with pOCT amino acids 1-141 or amino acids 1-242 to create PO-SA 141 or PO-SA 242, respec- tively (Fig. 1). The import of PO-SA 141 was compared with that of p0-14U, which is PO-SA 141 lacking the Tom7Op transmembrane segment. Both constructs contain the matrix signal sequence processing site, which occurs between amino acids 32 and 33 of the pOCT sequence (27). As shown in Fig. 2, A and B, the pattern of import of PO-SA 141 and pO-14lA into intact mitochondria was very similar to that of the control

A schematic of the various protein constructs employed in matrix protein, PO-DFIFR For all three constructs, sedimen- this investigation is presented in Fig. 1. pOMD29 contains the tation and processing was dependent on the presence of mito-

Relocating a Signal Anchor Se quence to the Inner Membrane 12059

- 4 + + + Mitos - - - + - Post-Trypsin - - - - + CCCP

10% 30 4 30 30 30 Temp {OC)

- + + + + Mitos - - - + + Alkali

10% 30 4 30 4 30 Temp (OC)

FIG. 2. Import of PO-SA 141, PO-141& and pO-DHFR into intact mitochondria. Stmdnrd import reactions and analyses were con- ducted as described under 'Materials and Methods." A, the 36S-precur- ror proteins, as indicated, were incubated with (lanes 3-61 or without (lane 2 ) mitochondria (Mitos) at either 4 (lane 3) or 30 " C (lanes 2 and 4-61, and in the presence (lane 6) or absence (lanes 2-51 of L.0 k\t CCCP. At the end of the import reaction, an additional incubation was conducted in the presence (lane 5 ) or absence (lanes 2-4 and 6 ) of trypsin (Post-Trypsin). Lane I . LO% of input precursor protein. B. lanes 1-4, ns in A. FoUowing import nt 4 (lane 5 ) or 30 " C (lane 61, mitochon- dria were recovered, and the alkdi-resistant fraction was obtained (Alkali). p and rn refer to precursor and processed (mnture) polypep tides, respectively, and are designated by arrows.

chondria (compare lane 2 with lane 4 ) and was temperature- sensitive (compare lane 3 with lane 4). The processed, but not the full-length precursor, forms of all three polypeptides were resistant to external protease (Fig. 2 4 lane 51, and the appear- ance of the processed molecules was dependent on the electro- chemical gradient across the inner membrane since it was abolished by CCCP (Fig. 2A, lane 6 ) . These results indicate that all three rnolecuIes were transported across the outer rnern- brane and that a t least their amino termini were located in the matrix compartment, which is the site of signal sequence cleav- age. Of particular note, however, is that the processed forms of the three polypeptides were extractable by alkali (Fig. 2B, lanes 5 and 6) indicating a failure to integrate into a membrane lipid bilayer. This suggests, but does not prove, that the polypeptides were translocated entirely to the matrix compart- ment. Identical results to those presented in Fig. 2, A and B, were obtained for PO-SA 242 and p0-242h (PO-SA 242 lacking the transmembrane region, Fig. 1) (data not shown) suggesting that the failure of PO-SA 141 and pOSA 242 to insert into

1 2 3 4 5 6 7 8 9

FIG. 3. Import of pO-SA 141, p0-1411, PO-DHFR, PO-SA 36, and pOMD29 into mitoplaets. Standard import reactions and analyses were conducted as described under 'hlaterials and Method$' and in Fig. 2 except thnt mitoplasts were used in place of intact mitochondria. The '"%-precursor proteins, as indicated ( p ) , were incubated in the presence (lanes 3-91 or absence (lane 2 ) of mitoplasts a t either 4 (lanes 3 and 8 ) or 30 ' C (lanes 2, 4-7 and 9 ) and in the presence (lanes 6 and 7) or absence (lanes 2-5. 8. and 9 ) of 1.0 &st CCCP. In lanes 5 m d 6 the mitoplasts were subjected to treatment with protease at the end of the import reaction (Post-Typsin), whereas the alkali-resistnnt fraction was analyzed in lanes 8 and 9 Wlkali). p and m refer to precursor and processed (mature) polypeptides, respectively. and ore designated by urroLus.

mitochondria1 membranes was unlikely to be the result of the immediate polypeptide context of the Tom70p transrnembrane segment.

To examine the possibility that the transmembrane portion of the Tom70p signal anchor in PO-SA 141 arrested import of the polypeptide across the inner membrane but failed to permit release from the translocation pore into the surrounding lipid bilayer, import of PO-SA 141 was examined in mitoplasts and compared with various control polypeptides. The generation of mitoplasts (32) was monitored by the release of the intermem- brane space marker, sulfrte oxidase, which was over 90% com- plete as judged by Western blot analysis (not shown). As shown in Fig. 3 (top panel), PO-SA 141 was imported and processed (lane 41, and the processed form of the molecule demonstrated A*-dependent resistance to exogenous trypsin (compare lanes 5 and 6 with lane 4 ) indicating complete translocation of the polypeptide chain to the soluble matrix compartment. Consist- ent with this conclusion, both PO-DHFR and PO-SA 141 were also protected from trypsin following import into intact mito- chondria and subsequent hypo-osmotic shock of the organelle to disrupt the outer membrane (20) (not shown). As expected (see also Fig. 21, the imported product in mitoplasts was ex- tracted by alkali (lane 9). Very similar import results were obtained for p0-141A and PO-DHFR (Fig. 3, panels 2 and 3) and for PO-SA 242 and PO-2426 (not shown). Finally, PO-SA 141 was completely degraded by trypsin following incubation with intact mitochondria in the presence of CCCP (not shown), indicating that PO-SA 141 did not insert into the outer mem- brane even in the absence of an electrochemical potential across the inner membrane.

Previous studies have documented import and insertion of pOMD29 into the inner membrane of mitoplasts in the Nin-COut orientation (3) (Fig. 58). As shown in Fig. 3, panel 5, this

Relocating a Signal Anchor Sequence to the Inner Membrane

- + + + + + M ~ O S - - - + + - Post-Twp~in - - - - - + Alkali - - - - + - CCCP

10% 30 4 30 30 30 30 Temp PC)

FIG. 4. Mutations in the yTom70p domain of PO-SA 141 that permit insertion into the mitochondrial inner membrane. -4, the nmino acid sequence (single letter codvr of the transmembrane portion of the yTom70p s i p d nnchor (nmino acids 11-29) is shown together with mutations of almine residues a t positions 14, 15, 17, and 18 to isoleu- cine. These mutations were made in PO-SA 141 to create PO-SA 141-14 and result in the predicted hydrophobic index of the Tom70p transmem- brnne domain incrensing from 1.17 to 1.74, as determined using the Secondary Structure Prediction. Prosis Program (Hitachi Software En- gineering Co., Ltd.). 8, standard import of PO-SA 141-14 and PO-DHFR into intact mitochondria was performed as described under "Materials and Methods." Conditions and analyses were the same as in Fig. 2. oa is the nomenclature.

results in acquisition of resistance to extraction by alkali (com- pare lanes -I and 9) but leaves the bulk of the polypeptide exposed a t the surface of mitoplasts where it is susceptible to degradation by exogenous t&sin (compare lanes 4-and 5). Thus, the transmembrane segment of Tom70p is competent for insertion into the inner membrane in the context of pOMD29 but not in the context of PO-SA 141 or pO-SA 242. However, of particuiar interest were the findings with PO-SA 36, in which the Tom7Op transmembrane segment is immediately adjacent to the pOCT matrix-targeting signal. Import and processing of PO-SA 36 (Fig. 3, panel 4) was dependent on the presence of mitoplasts (compare lanes 2 and 4 with an intact electrochem- ical potential (compare lanes 4 and 7), indicating that the Mi, terminus of the polypeptide reached the matrix. The bulk of the polypeptide, however, was accessible to trypsin (lane 5 ) and therefore was located outside the organelle. This is in distinct contrast to PO-SA 141, p0-141& and PO-DHF'R, for which the processed forms of the polypeptides-were protected against trypsin (panels 13, compare lanes 4 and 5). In addition, and in contrast to pOMD29, the processed form of PO-SA 36 was extractable by alkali (compare lanes 4 and 9). Taken together, therefore, these results suggest that the Tom7Op signal anchor transmembrane segment caused PO-SA 36 to pause or arrest during translocation across the inner membrane, but it did not trigger release of the transmembrane segment into the sur- rounding lipid bilayer.

Increasing the Hydrophobicity of the Torn7Op Signal Anchor Tmnsmembrane Segment Permits Insertion into the Inner Membrane-In a previous study (221, mutations were intro- duced into the transmembrane segment of the Tom70p signal anchor, in which alanines a t positions 14. 15, 17, and 18 were converted to isoleucine. These changes did not affect the ability of the signal anchor in the context of pOMD29 to select and

A Mitochondria

*DN 36

Mitoplasts

FIG. 5. Schematic of the location and orientation of the vari- ous hreion proteins following import into mitochondria (A) and mitoplasts a). See text for discusciion. Cyto, cytosol; OM, outer mem- brane; IMS, intermembrane space; IM, inner membrnne; N, NH, ter- minus: ytipplrd ouals. inner membrane translocation machinery; black cylinder, Tom7Op signal mchor; hatched cylinder, Tom70p signnl an- chor with four alimines mutated to isoleucines which increases its hydrophobicity. Processing of the NH, terminus in the matrix is shown.

insert into the mitochondria1 outer membrane in uitm (22). Here, the identical changes were introduced into the Tom70p hydrophobic domain of pOSA 141, to create pOSA 141-14 (Fig. U). They resulted in the mean hydrophobicity of this segment increasing from 1.17 to 1.74 (Fig. 4A) employing the hydropho- bicity scale of Kyte and Doolittle (36) (Secondary Structure Prediction, Prosis Program, Hi tachi Software Engineering Co., Ltd.). Import and processing of PO-SA 141-14 was dependent on mitochondria (Fig. 48, compare lanes 2 and 41, and the pro- cessed product, but not the full-length precursor. acquired re- sistance to external trypsin (lane 5 ) that was dependent on the presence of an electrochemical gradient (lane 6). Thus, the trypsin-resistant component of pOSA 141-14 had crossed the outer membrane and deposited its NH, terminus into the ma- trix. However, in contrast to PO-SA 141 (Figs. 2 and 3) and PO-DHFR (Figs. 2,3, and the lower panel in Fig. 48) that are imported to the matrix where they remain extractable by al- kali, pOSA 141-14 was resistant to alkaline extraction (Fig. 4B, compare lanes 4 and 7). Therefore, the results show that PO-SA 141-14 was inserted into the inner membrane. Since the

Relocating a Signal Anchor Sequence to the Inner Membrane 12061

polypeptide has a single downstream transmembrane segment, t b means that this bitopic-processed polypeptide spans the inner membrane once, leaving the COOH terminus in the in- termembrane space. This was confirmed following import into mitoplasts, where subsequent digestion of processed PO-SA 141-14 by trypsin yielded a polypeptide fragment whose size was consistent with an N,,-C,,, orientation (data not shown).

Conclusions-in this study, we have examined the Function of the Tom70p signal anchor transmembrane segment when placed in different contexts relative to a matrix-targeting sig- nal. The results are summarized in Fig. 5. When the Tom7Op transmembrane segment is contiguous to the pOCT matrix- targeting signal (PO-SA 36) and presented to intact rnitochon- dria in oitro, it inserts into the outer membrane in the N,,-C,, orientation, which is opposite that observed for the native Tom70p signal anchor (pOMD29) (Fig. 5A). When this same polypeptide construct, PO-SA 36, is presented to rnitoplasts, the transmembrane segment causes arrest of the polypeptide across the inner membrane in the N,,-C,,, orientation, but it does not trigger insertion into the membrane lipid bilayer (Fig. 5B). If placed at some distance downstream of the matrix- targeting signal (PO-SA 141 and PO-SA 242) and presented to either intact mitochondria or mitoplasts, the transmembrane segment is no longer capable of arresting transIocation or trig- gering insertion into either mitochondria1 membrane, and the protein is translocated entirely to the matrix compartment. Increasing the net hydrophobicity of the Tom70p transmem- brane segment within the context of this latter construct, how- ever, results in its insertion into the inner membrane in the Ni,-C,,, orientation (PO-SA 141-14). These findings are similar to those found for bacteria, where it has been shown that protein insertion into the cell membrane requires a threshold hydrophobicity for the transmembrane segment (37).

The observed outcomes that were specified by the hydropho- bic domain within the various polypeptide constructs examined in this study closely mimic those specified by hydrophobic domains that exist within native proteins: signal anchor se- quences that direct insertion into the outer membrane, stop- transfer sequences that specify insertion into the outer or inner membrane, and intermembrane sorting sequences that cause translocation arrest across the inner membrane. The results, therefore, help to identify characteristics of these closely re- lated hydrophobic segments that are likely important for func- tion. In particular, our findings suggest that insertion of a potential transmembrane segment into the inner membrane requires a reIatively high net hydrophobicity when this seg- ment is located at some distance downstream of a strong ma- trix-targeting sequence. In the absence of an outer membrane (i.e. mitoplasts), however, a transmembrane segment of lower hydrophobicity will either insert into the inner membrane if located adjacent to a weak matrix-targeting signal (i.e. the native Tom70p signal anchor) or it will only arrest transloca- tion if adjacent to a strong matrix-targeting signal. The ability of a hydrophobic domain to insert into the inner membrane, therefore, likely depends on four inter-related factors: 1) its net hydrophobicity, 2) whether or not it is permitted to pass across the outer membrane, 3) its distance from a matrix-targeting

signal, and 4) the relative strength of the matrix-targeting signal. How the constituent components of the outer and inner membrane translocation machineries discriminate between these different contexts and control protein sorting, however, is not known. Presumably, it involve^ a complex interplay be- tween the dynamic and reversible interactions that can occur between the two import machineries and, in addition, may result from the different requirements that individual precur- sor proteins may have for ATP, the electrochemical potential, and chaperone interactions.

Acknowledgment-We are grateful to Dr. J. Orlowski for critically reading the manuscript.

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CHAPTER 4:

Mitochondria1 Cytochrome c Oxidase Subunit IV is Phosphorylated by an

Endogenous Kinase.

ABSTRACT:

This study was undertaken to identify novel mitochondria1 membrane proteins that are

potential targets for phosphorylation. Mitochondria1 membranes were incubated in the

presence of [ y 3 2 ~ ] - ~ ~ ~ and the Triton X- 1 14 extractable protein was subjected to ion-

exchange and polyacryiamide gel chromatography to purify a major phosphorylated protein

of approximately 17,000 Da. The determined peptide sequence of the purified

phosphoprotein corresponded to a segment of cytochrorne c oxidase subunit IV. an inner

membrane protein of 17,160 Da. The identity of the phosphoprotein was confirmed by

two-dimensional electrophoresis and Western blotting. The results identify mitochondria1

cytochrome c oxidase subunit IV as a protein which is phosphorylated by an endogenous

ki nase.

1. Introduction

The nuclear genome encodes the majority of proteins which reside in mitochondria

[l]. In order for eukaryotic cells to function properly, mitochondria must communicate

with the nucleus to regulate the synthesis and assembly of the mitochondria1 proteins [2].

These proteins are translated in the cytosol and then targeted, imported and sorted into their

final location within one of four compartments; the outer or inner membrane, the

intermembrane space or the matrix (for reviews see [3-51).

Communication between and within cells is often achieved through signal

tmnsduction pathways (for reviews see [6, 71). These signals can be transduced through

enzymatic phosphorylation and dephosphorylation of proteins in a sequential cascade [8]

which reversibly alters their structural and functional properties. Thus it is reasonable to

suspect that communication between the nucleus and the mitochondria may involve one or

more protein kinase, which along with some of their target proteins may reside within

mitochondria.

Indeed, i t has been shown that the regulation of mitochondrial pyruvate

dehydrogenase [9] and branched-chain a-ketoacid dehydrogenase 1101 involves

phosphorylation and their distinct kinases have been cloned from rat heart [11, 121. The

presence of other kinases within mitochondria has also been reported. These include

c AMP-dependent protein kinase [13- 171, CAMP-independent protein kinase [ 18-20],

tyrosine kinase [21. 221, protamine kinase [23], phosphorylase kinase (in brain) [24] and

casein kinase I1 [17, 231. Phosphorylated proteins within mitochondria from mammals,

yeast and plants have been examined by several investigators. To date, however, only

three proteins have been routinely identified: the a-subunit of pyruvate dehydrogenase [9],

the a -subunit of the branched-chain a-ketoacid dehydrogenase [lo] and the

autophosphorylated subunit of succinyl-CoA synthetase 1251. A limited number of other

candidate phosphoproteins exists [13, 22, 26-34], but very few of these have been

identified 135, 361.

Here, we report the purification of a 17,160 Da mitochondria1 membrane protein

which is phosphorylated by an endogenous kinase. Amino acid sequencing and

immunological analysis revealed that it is cytochrome c oxidase subunit IV, an inner

rnitochondrial membrane protein.

2. Materials and Methods

General Procedures - Previous articles describe the routine procedures used in this

study [37, 38; and references cited therein]. These include purification of mitochondria

from rat heart and analysis of proteins by SDS-PAGE and fluorography. All preparative

procedures and centrifugation were performed at 40C.

Preparation of microsomes, mitochondria and mitochondria1 rnembmnes - Dog

pancreas microsomes were isolated as previously described [39] and were kindly provided

by Dr. J.J.M. Bergeron's laboratory (McGill University). Liver mitochondria1 membranes

were prepared by a modified version of a previously described method [30]. Livers were

obtained from six 200-250 g Sprague-Dawley rats and were minced and washed in ice-

cold 0.25 M sucrose. The minced liver was then homogenized in a 10-fold volume of 0.25

M sucrose in a motorized Potter-Elvehjem homogenizer using eight up and down cycles at

500 rpm. The homogenate was diluted with 0.25 M sucrose to a final volume of 240 ml

and centrifuged at 750g for 10 min. The supernatant was recovered and centrifuged at

7000g for 10 min. The resulting pellets were resuspended in 50 rnl of 0.15 M sucrose and

hand homogenized with five up and down passes. The homogenate was diluted with 0.25

M sucrose to 210 ml and centrifuged at 7000g for 10 min. The resulting pellets

(mitochondria) were washed by repeating the previous step. To obtain mitochondria1

membranes, the mitochondria were resuspended in 60 rnl of 10 mM Tris-POq, pH 7.5 and

incubated on ice for 10 rnin, followed by the addition of 16 ml of 2 M sucrose, 2 ml of 40

rnM MgS04 and 2 ml of 10 mM ATP and the incubation continued for 10 min. Aliquots

(20 ml) were sonicated for 20 sec using a Sonic Dismembrator (ARTEK Systems Corp.,

NY, USA) with a small probe, set at 60. Aliquots (10 rnl) of the sonicate were layered

onto 12 rnl of 1.2 M sucrose and centrifuged at 100,000g for 60 min. The interface

between the two fluid layers was collected and pooled and the total volume was brought up

to 200 ml with 20 ml of 1.5 M NaCI, 0.5 M Tris-HCl, pH 8.0 and distilled water. The

diluted interface was centrifuged at 100,000g for 60 min. The pellets were recovered and

resuspended in 2 ml of 0.25 M sucrose and the protein concentration was determined (Bio-

Rad Protein Assay). The resuspension was centrifuged for 35 min at 130,000g and the

pellets were resuspended at a concentration of 10 m g h l in 20 rnM Hepes, pH 7.4, 50%

glycerol and 1 mM dithiothreitol and stored at -700C.

Phosphorylntion of rnicrosornes, mitochondria nrtd mitockondrial membranes -

Membranes ( 10-35 mg) were diluted to 4 mp/ml with 20 rnM Hepes. pH 7.4 and 10% was

incubated with y 3 2 ~ - ~ ~ ~ diluted with non-radioactive ATP to give a concentration of 28

pM and an activity of 1 pCifp1; the remaining 90% was incubated with non-r~diolabelled

ATP (28 pM) using a scaled up version of the method of Wada et nl. [4 11. The reaction

was incubated and stopped as in Rindress er nl. [3 11. In addition to the stop solution, 2.5

m M P-glycerophosphate was added to the terminated reaction.

Purijkcition oj9ppl7 - The phosphorylated membmnes were extracted with Triton

X- 1 14 (Tx- 1 14) I using the method of Bordier [42]. Condensed Tx- 1 14 was a generous

gift of the laboratory of Dr. J.J.M. Bergernn (McGill University). The detergent phase

was subjected to anion exchange chromatography using DEAE-Sepharose Fast Flow

(Pharmacia LKB Biotechnology Inc.) equilibrated with Buffer A (20 m M Tris. pH 7.4 and

0.2% Tween 30), washed with 0.01 M NaCl in Buffer A and eluted with a gradient of

0.01-0.5 M NaCl in Buffer A containing 2.5 mM P-glycerophosphate and 5% glycerol.

The collected fractions were monitored for protein (A280) and radioactivity. Fractions

( 136- 158) containing the peak radioactivity were pooled and concentrated by dialysis

versus Buffer A containing 2.5 m M P-glycerophosphate and 60% glycerol. The sample

was precipitated with ethanoUhexanes (4: 1 ), resuspended in 0.5 ml SDS-sample buffer,

incubated at 600C for 10 min and loaded onto a Model 491 Prep Cell (Bio-Rad

Laboratories) and subjected to preparative gel electrophoresis. The Prep Cell contained a 6

cm separating gel (16% acrylamide) and a 1 cm stacking gel (4 % acrylamide) in the 28 rnm

diameter tube. The electrophoresis was performed at 40 rnA for 2.5 h until the dye front

started to elute and 0.5 ml fractions were collected for another 2 h. The elution buffer

contained 0.02% SDS and flowed at 0.1 ml/min. The eluted fractions were monitored for

radioactivity and the peak fractions were analyzed by SDS-PAGE and autoradiography.

The peak fractions ( 16- 19) were precipitated with ethanollhexanes and resuspended in

SDS-sample buffer, subjected to SDS-PAGE and electroblotted to nitrocellulosr

membrane.

Identification o f p p l 7 - The blots were visualized using 0.2% Ponceau S stain and

autoradiography and the band corresponding to pp 17 was excised from the blot. The

protein on the blot was subjected to uypsin digestion and the sequence of the resulting

peptides was determined (Harvard Microchemistry Facility, Cambridge, MA, USA).

Two-dimensional (2D)-gel elrctruphoresis - 25 pg of phosphorylated rat heart

mitochondria1 membranes (0.5 mg/ml) were loaded onto the acidic end of an

isoelectricfocusing gel (non-equilibrium pH gradient electrophoresis) [43] containing 2%

ampholytes (Bio-Lyte 3- 10) and subjected to 700 v for 1 h. The protocols and procedures

were as provided by Bio-Rad except for the changes which are noted above. Proteins were

separated in the second dimension by 12% SDS-PAGE and analyzed either by Western

blotting or by staining [JJ] and subsequent autoradiography or phosphorescent imaging.

Anti-COX IV antibodies were a generous gift from Prof. Dr. B. Kadenbach (Philipps-

Universitiit Marburg).

3. Results and Discussion

Rat heart mitochondria were purified and constituent protein substrates

phosphorylated by an endogenous lunase, using a ratio of 10% y 3 2 ~ - ~ ~ ~ and 90%

nonradiolabelled ATP. The resulting phosphorylation pattern is shown in Fig. I , which

reveals the existence of a limited number of phosphoproteins in mitochondria (Mito, Lane

T) compared to microsomes (Lane TI. These proteins were partitioned into either the

aqueous (Lane A) or detergent (Lane D) phase using Tx-114. Several phosphorylated

proteins were detected in the total mitochondria1 fraction (Lane T). One, with an

approximate molecular mass of 43,000 Da most likely corresponds to the a subunit of the

E 1 component of pyruvate dehydrogenase as previously reported [lo] and it partitions into

the aqueous phase as expected (Lane A). A second has a molecular mass of approximately

37,000 Da and likely corresponds to succinyl-CoA synthase since it is acid labile (the band

is lost with acidic gel staining, not shown) [25] and is no longer visible in either the

detergent or aqueous phase after detergent extraction with Tx-114. A third

phosphoprotein, with an approximate molecular mass of 17,000 Da (pp17), is partitioned

into the detergent phase (Lane D compared to Lane T and Lane A). This protein was absent

in rnicrosomes (Lane D). Taken together, these data suggested that pp17 is an integral

mitochondria1 membrane protein. It was further characterized in order to determine its

molecular identity.

As a first step, the total mitochondria1 membrane fraction was obtained from rat

liver and phosphorylated as described under "Materials and Methods" (Fig. I, MM, Lane

T) yielding many more phosphorylated proteins compared to the situation with intact

mitochondria. The major phosphorylated protein in the membranes was pp 17. Again,

when the membrane fraction was subjected to Tx-114 extraction, pp17 partitioned in the

detergent phase (Lane D compared to Lane T and Lane A).

FIG. I . Pattern of y 3 2 ~ - p h o s p h o r y l a t e d proteins in microsomes,

mitochondria and mitochondrial membranes. Endogenous phosphory lation with

@ZP] ATP of dog pancreas microsomes, rat heart mitochondria (Mito) or rat liver

mitochondria1 membranes (MM) was as described under "Materials and Methods". A 17

kDa phosphorylated protein is enriched in mitochondrial membranes and is resistant to Tx-

114 extraction, its mobility is indicated by the mow on the right (pp17). Molecular mass

markers are visible in lanes S and their size (in kDa) is indicated on the left. Approximately

10 pg of microsoma1 protein was loaded in lane T and a total of 40 pg was loaded in D and

A. For mitochondria and MM, 20 pg was loaded in lanes T and a total of 80 yg was

loaded in lanes D and A. T, total fraction; D, detergent Tx-114 fraction; A, aqueous

fraction; S, standards; DF, dye front.

S L T D* A S I T D A" T D A l

Microsomes Mito MM

F I G . 2. Purification of pp17: DEAE-Sepharose chromatography.

Mitochondria1 membranes were phosphorylated with [y32~] ATP and extracted with Tx-

1 14. The detergent fraction after Tx-114 treatment was applied to a DEAE-Sepharcxe Fast

Flow coIumn as described under "Materials and Methods". A, Column fractions were

monitored for their protein content (A280) and radioactivity (CPM) profile. B, Coomassie

blue-stained protein profiles of total mitochondrial membranes (MM) and the fractions from

the peak of protein and radioactivity from the column. C, Autoradiographic profile of the

peak column fractions shown in B. The location of pp 17 is indicated with an arrow on the

left. Molecular mass markers are indicated on the right (in kDa). DF, dye front; Bmcket

indicates the peak fractions which were pooled (136- 158).

Fraction Number

Purification

phosphorylated and

o f p p l 7 - In

the proteins

order to purify

that partitioned

applied to a DEAE-Sepharose Fast Flow column.

39

pp 17, mitochondrial membranes were

into the Tx-114 detergent phase were

Most of the protein did not bind to this

column and was eluted in the flow through. A small amount of bound protein eluted as a

single peak (0.2-0.3M NaC1) and the protein peak coincided with the peak of radioactivity

(Fig. 2, Panel A). The Coomassie blue stained protein profile of the mitochondrial

membranes (MM) and the peak proteidradioactive fractions is shown in Fig. 2. Panel B.

pp17 was greatly enriched in the peak fractions as compared with the mitochondrial

membranes (MM). The autoradiographic profile of the peak fractions is shown in Fig. 2,

Panel C. These fractions were pooled and applied to a preparative electrophoresis

polyacrylamide gel. One major peak of radioactivity was recovered and eluted in fractions

15-19 (Fig. 3, Panel A). The Coomassie blue stained protein and radioactivity prodles of

these peak fracrions and of the original Prep Cell load are presented in Fig. 3, Panels B and

C, respectively. The lower band of the doublet seen on the Coomassie blue protein gel

corresponds with the radioactivity. The peak fractions containing the lower band were

pooled and concentrated and subjected to SDS-PAGE.

Identification of pp l7 - Following SDS-PAGE, proteins were electroblotted to

nitrocellulose and the radioactive band corresponding to pp17 was excised. It was

subjected to trypsin digestion and protein sequencing of a constituent peptide. The ten

amino acid sequence which was obtained showed a perfect match with amino acids 150-

159 of rat liver cytochrome c oxidase subunit IV (COX [V).

To confirm the identity of pp17 as COX IV, ZD-gel electrophoresis followed by

Western blotting was performed on phosphorylated whole mitochondria from rat heart.

The Coomassie blue stained protein profile of the 2D-gel is shown in Fig. 5, Panel A. The

17,000 Da phosphorylated protein on the phosphorescent image of the 2D stained gel

(Panel B, arrow) has the same localization as COX IV on the Western blot of the 2D-gel

F I G . 3. Purification of pp17: Preparatory gel electrophoresis. The peak

fractions from the DEAE-Sepharose column were pooled, dialyzed and concentrated and

applied to a preparatory polyacrylamide gel electrophoresis column as described under

"Materials and Methods". A , Radioactive (CPM) profile of the eluted fractions. B ,

Coornassie blue-stained protein profiles of the preparatory cell load (Load) and the peak

radioactive fractions. C, Autoradiographic profile of the load and column fractions shown

in B. pp 17 is indicated with an arrow on the left, molecular mass markers are indicated on

the right (in kDa). DF, dye Front; Bracket indicates the peak radioactive fractions.

Fraction Number

FIG. 4. Comparison of trypsin generated peptide sequence with cytochrome

c oxidase subunit IV. Amino acid sequence of rat cytochrome c oxidase subunit IV

(Gopdan er al., 1989; Goto er ol., 1989), with the peptide obtained from trypsin digestion

and sequencing, shown in bold. Several potential phosphorylation consensus sites are

underlined, I and la are protein kinase c, 2 is casein kinase 11, 3 is CAM kinase I1 or

CAMP dependent kinase, 4 is glycogen synthase kinase-3 and 5 is casein kinnse I (1 and 2

were identified by Prosite; la, 3, 4 and 5 were identified from Kennelly and Krebs

( 199 1)). Bold numbers indicate amino acid position relative to initiator methionine, which

is 1. Arrow, the site of cleavage in the signal sequence where processing would otherwise

take place in the matrix. The predicted transmembrane domain is boxed (Tsukihara et nl..

1996). The single letter amino acid code is used. + potential sites of phosphorylation for

consensus sites 5 or la at position 72; * potential sites of phosphorylation for the other

consensus sites,

+ + * - - - - t * MLATRALSLIGKRAISTSVCLWGSVVKSEDYALPSYVD

1 3 35 la - 2 40 4 or 5 * + *

RRDYPLPDVAHVKLLSASQKALKEKEKADWSSLSRDEKVQ - - 41 1 1 a 2 80 -

VNPIQGFSAK . . . . . . . .

DYNKNEWKK 161 169

(Panel C, arrow). Thus, COX IV is phosphorylated and corresponds to pp17 in rat hem

mitochondria.

Conchisions - We have identified COX IV as the phosphorylation product of an

endogenous kinase whose activity is expressed in both whole mitochondria and in isolated

mitochondria1 membranes. Cytochrome c oxidase (COX) is the terminal enzyme complex

of the mitochondrial respiratory chain and catalyzes the reduction of molecular oxygen to

water coupled to the translocation of protons across the inner mitochondrial membrane (for

review see [45]). In mammalian mitochondria, COX is composed of ten nuclear encoded

subunits and three mitochondrial encoded subunits [46]. Subunit IV is a nuclear encoded

subunit whose crystallographic structure resembles a dumbbell [47]. It contains one

transmembrane domain and resides in the inner membrane with the N-terminus facing the

matrix and the C-terminus in the intermembrane space. It has been proposed that the

nuclear encoded subunits of COX affect the catalytic function of the mitochondrial encoded

subunits by binding allosteric effectors such as substrates, cofactors, ions, nuleotides and

hormones [48]. Specifically, ATP has been shown to bind to two subunits of bovine hem

COX, including subunit IV [49] and to six or seven subunits of COX from bovine liver or

hem, respectively, including subunit IV [50]. As well, there is evidence indicating that the

yeast homologue of subunit IV, subunit V. may regulate catalysis and modulate the

function of the holoenzyme [5 11. Also, it has been proposed that subunit IV functions as a

transmitter of signals, such as channeling protons into the proton translocating pathway

There are many potential phosphorylation consensus sites for several different

kinases within the mature sequence of COX IV as well as in the signal sequence (Fig. 4).

Some of these consensus sites are located within evolutionary conserved regions (amino

acids 26-32 and 54-84), [53], implicating them as good candidate sites for

phosphorylation. The conserved region of amino acids 26-32, besides containing several

phosphorylation consensus sites, is also the region identified as important in translocating

FIG. 5. Identification of ppl7 as cytochrome c oxidase subunit IV. Rat heart

mitochondria were phosphory lated as described under "Materials and Methods" and loaded

onto the acidic end of an isoelectric focusing gel (non-equilibrium pH gradient

electrophoresis). The proteins were then separated in the second dimension by 12% SDS-

PAGE and analyzed by autoradiography, phosphorescent imaging and Western blotting.

A, Coomassie blue-stained profile of the protein pattern on the ZD-gel. B, Phosphorescent

image of the gel in A after is was analyzed using a Fuji BAS-2000 Bio-Image Analyzer.

The migration of pp17 is indicated with an arrow. C, Western blot of a 2D-gel

electroblotted to nitrocellulose and probed with mouse anti-cytochrome c oxidase subunit

IV antibody. The position of cytochromr c oxidase subunit IV is indicated with an arrow.

Molecular mass markers are indicated on the left (in kDa). The direction of the first and

second dimensions are indicated by the arrows on the top and side of the gels. DF, dye

front.

Acidic Basic

Acidic Basic

Acidic Basic

SDS-PAGE

I

SDS-PAGE

t

protons across the membrane [ X I . While speculative, it is possible that the translocation

of protons could be regulated by phosphorylation.

Several candidate endogenous kinases in mitochondria have been identified,

including CAMP-dependent, CAMP-independent, tyrosine, protarnine and phosphorylase

kinases, and casein kinase 11. It is noteworthy that one of these is casein kinase I1 [17, 231

since there are several consensus sites for casein kinase I1 located within the COX IV

sequence. including one within the conserved region of amino acids 54-81. The presence

of phosphorylation consensus sites within the presequence may be relevant to import of the

protein into the mitochondria, as was shown recently for chloroplast precursor protein

transit sequences [54].

An 18 kDa protein of Complex I(18 kDA (IP) AQDQ) has also been shown to be

phosphorylatrd in bovine heart mitochondria [36] indicating that other proteins of the

respiratory complexes may be regulated by phosphorylation as well.

The site and significance of the phosphorylation of COX IV is not known at this

time. It is reasonable to assume, however, that it may contribute to the signalling pathway

which regulates COX IV, and may play a key role in modulating COX activity and

mitochondrid function.

Acknowledgment - We are grateful to Pamela H. Cameron for performing the experiment

shown in Fig. 1 and to Drs. W.E. Mushynski and J. Orlowski for critically reading the

manuscript.

l ~ h e abbreviations used are: Tx- 1 14, Triton X- 1 14; 2D, two-dimensional; COX IV.

cytochrome c oxidase subunit IV; COX, cytochrome c oxidase.

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CHAPTER 5:

General Discussion.

5.1 Summary

To understand organelle biogenesis, one needs to know which topogenic sequences

within preproteins are involved and how these sequences specify orientation, arrest and

insertion within, and sorting between membranes. Therefore, one needs to understand not

only the function of these signals, but also which component(s) of the Tom and Tim they

interact with. The work in this thesis has examined this problem by investigating different

topogenic sequences within precursor proteins and their effects on the targeting of these

proteins. Another area which is not well understood is how the mitochondrion and nucleus

communicate with each other to regulate protein synthesis and trafficking to the

mitochondrion. Since signal transduction is a common method in the cell for

communication, the identification of mitocbondrial phosphoproteins is the first step in

elucidating what signal transduction pathways may exist to and from the mitochondrion.

More specifically, my research has investigated the roles of signal-anchor

sequences, alone or in combination with MTS's, in targeting to and sorting within the

mitochondria, as determined by their locations within preproteins. The orientation of a

signal-anchor in the OM was found to be dependent on the amphiphilicity of the

hydrophilic region of the signal-anchor and was not due to the number and position of

positively-charged amino acids. The amphiphilic region has lipid-binding properties which

could contribute to its retention on the OM surface. As well, the signal-anchor

transmembrane region, when placed downstream of a MTS was unable to function as a

stop-transfer sequence in the IM. However, if the hydrophobicity of this region is

increased, the region will now arrest and insert into the IM. Therefore, the ability of

hydrophobic regions to insert into the IM is dependent on four inter-related factors: I ) its

net hydrophobicity, 2) whether or not it is permitted to cross the OM, 3) its distance from a

MTS and 4) the relative strength of the MTS. Last, it was also determined that COX IV is

phosphorylated by an endogenous b a s e which is present in both whole mitochondria and

in preparations of isolated membranes. This suppons a role for protein kinases and

phosphoproteins in mitochondria1 function, although the physiological importance of this

process remains to be determined.

5.2 Orientation of Signal-Anchor Sequences

The yeast OM receptor, yTorn70p, contains a signal-anchor sequence at its NHz-

terminus. This sequence targets and inserts the protein into the OM of mitochondria in an

Nin-Ccyto orientation. The signal-anchor is composed of two regions. the NH2-terminal

hydrophilic positively charged region (aa 1-10) and the predicted hydrophobic

transmembrane region (aa 11-29). In addition to targeting and insertion information, the

signal-anchor can also specify the orientation of the protein within the OM. If the NHz-

terminal hydrophilic region is replaced by a MTS, the signal-anchor will now be retained

on the OM surface and the protein will adopt an Ncyto-Gin orientation (Li and Shore,

19921). The same orientation is found for a MTS sequence contiguous to a heterologous

stop-transfer sequence (Nguyen et al., 1988). I determined that the number and position of

the positively charged amino acids within the hydrophilic region, did not affect the

orientation of the signal-anchor in the OM. Instead, the retention of the signal-anchor on

the OM was due to the amphiphilic nature of the NH2-terminal hydrophilic region.

Reducing the amphiphilicity of this region will cause the reversal of orientation to Nin-

Ccyto (Sternam et czl. , 1996). This is unlike ER proteins, where the net charge flanking

the hydrophobic region specifies the orientation of the protein (von Heijne and Gavel,

1988; Sipos and von Heijne, 1993). or bacterial proteins where the number of positively

charged amino acids specify the orientation of the protein, such that the positively charged

regions iue retained in the cytosol (von Heijne, 1986; Gafvelin and von Heijne, 1994). It

would be interesting to determine whether the orientation of other mitochondria1 OM

proteins containing either a NHz-terminal or COOH-terminal signal-anchor, such as

Tom20 (Goping et al., 1995) or Bcl-2 (Nguyen et al., 1993), respectively, could also be

reversed by altering the amphiphilicity of the hydrophilic regions of their signal-anchors.

The amphiphilic region also has greater lipid-binding characteristics than a reduced

arnphiphilic mutant implying a role for binding to the membrane surface in the retention of

the signal-anchor.

The role of the mitochondria1 OM receptors in the retention of the signal-anchor

sequence was not investigated in this study. One would expect that the protein receptors

would also interact with the NH2-terminal region of the protein and play a role in binding.

Therefore. i t would be interesting to examine the binding of the PO-OMD (the fusion

protein consisting of the pOCT MTS contiguous to the transmembrane region of the

yTom70p signal-anchor fused to DHFR) signal-anchor as well as that of the mutant with

reduced amphiphilicity to isolated receptors. Binding studies of precursor proteins have

been done with the cytosolic region of the Tom 70. Tom20 and Tom22 receptors

(Schlossrnann ct czl., 1994; Brix et al., 1997; Komiya et al., 1997; Schleiff et nl, 1997a;

1997b). All of the receptors can bind precursors independently of each other, but with

different specificities. Only Tom20 and Tom22 bind presequences in a manner which can

be competed with a synthetic presequence (Brix et d., 1997). Since Tom20 has been

shown to bind the MTS of pOCT in an amphiphilic a-helical state (Schleiff and Turnbull.

1997), one would expect that PO-OMD would also bind, since it contains the pOCT MTS

at its NH2-terminus. Whether the reduced amphiphilic mutant. which contains a mutant

pOCT MTS, would still bind or if it now binds to another region of Tom20 could also be

determined.

Previous studies have shown that pOMD29 (the fusion protein consisting of

yTom70p signal-anchor fused to DHFR) is able to insert into the IM in the absence of OM,

i .r . , in mitoplasts (Li and Shore, 1992b), but as seen in Chapter 3, PO-OMD arrests

translocation but does not insen into the IM. Hence, further characterization of the

targeting and sorting of the proteins studied in Chapter 2 in the absence of an OM, that is. if

they are presented directly to the IM, would elucidate the function of the different features

(charge, length, amphiphilicity) of the signal-anc hor in sorting. Would the charged-

reduced pO-OMD mutants be arrested or would they now be inserted into the IM? Are the

length-increased mutants of pOMD29 inserted in the IM? Would all of the mutants still

behave as their "parent" proteins Like they do in the OM? What role does the arnphiphilicity

of the hydrophilic region play at the IM? Does this region still bind to lipids or would it

interact with the Tim components, perhaps with Tirn23? What sons of interactions, if any,

would occur with the reduced amphiphilic mutant? It is possible that IM- and matrix-

targeted proteins which contain different types of signal sequences. may use different

components of the Tim during translocation. Therefore, it would also be of interest to

determine if pOMD29, which contains a signal-anchor, and PO-OMD, which contains a

MTS within its signal-anchor, use the same or different Tim components. One could use

crosslinking and yeast deletion mutants to answer these questions.

5.3 Inner Membrane Targeting

Mitochondria1 IM targeting signals for bitopic proteins consist of a NH2-terminal

MTS combined with a hydrophobic stop-transfer region downstream (Glaser ct al., 1990).

The stop-transfer region arrests translocation and inserts the protein into the IM with a Nin

orientation. A protein which targets to the IM was also created by placing a heterologous

stop-transfer sequence downstream of the MTS in pOCT (Nguyen and Shore. 1987).

The transmembrane region of the yTom70p signal-anchor was also examined to

determine how it functions in targeting when it is placed in different contexts relative to a

MTS. It was determined that the signal-anchor transmembrane region placed contiguous

with the MTS will insert with the reverse orientation to the native yTom70p signal-anchor,

Ncyto-Gin in the OM. However, when this construct is presented to mitoplasts, the

transmembrane region will cause translocation arrest across the IM, but it does not insert

into the LM. If the transmembrane region of the signal-anchor is placed downstream of the

MTS, it does not function as a stoptransfer for the IM and translocates through to the

matrix. However, if the hydrophobicity of the yTom70p transmembrane segment is

increased within this construct, it now inserts into the IM with a Nin-Cout orientation

(Steenaart and Shore, 1997a). In bacteria it has dso been determined that protein insertion

into the cell membrane requires a threshold hydrophobicity for the transmembrane segment

(Whitley et al.. 1995). These studies help to elucidate how closely related hydrophobic

sequences function differently in sorting, that is, as signal-anchor sequences which insert

into the OM, as stop-transfer sequences which specify insertion into the OM or IM, or as

translocation arrest signals for the IM which are found in IMS sorting sequences.

The question of how hydrophobic regions destined for the IM avoid insertion into

the OM is still not answered. The theory that once the Tim is engaged by the translocating

preprotein, the Tom is no longer able to recognize and insert a hydrophobic region is still

valid (Nguyen rr d., 1988). The identification of hydrophobicity as an important

characteristic of a stop-transfer sequence downstream of a strong MTS, may shed some

light on how the OM and LM distinguish between different sorting signals, inserting some

or allowing others to pass through. The Tom and Tim both recognize and insert the signal-

anchor of yTom70p, however, if a MTS is inserted upstream of the transmembrane region

of the signal-anchor, neither the Tom or the Tim will recognize or insert the transmembrane

region. Therefore the presence of a MTS somehow prevents the Tom and Tim components

from inserting the transmembrane region into the membrane. If the MTS is placed

contiguous to the transmembrane region of the signal-anchor ( PO-OMD), the Tom will

insert the preprotein, although with an opposite orientation to that of the native signal-

anchor (pOMD29) and the Tim will arrest pOMD29's translocation but won't insert it. It is

possible that a different Tim complex is used for translocation when a MTS is present in the

preprotein. Most preproteins with an MTS use the Tim23-Tim17-Tim44 complex while

Tim1 1 functions for cytochrome b2, which has a hydrophobic region following its MTS

(IMS sorting signal) (Tokatlidis et al., 1996). Therefore, an increased hydrophobicity may

be needed to arrest proteins which are translocated through Tim23-Tim17-Tim44 versus

those which translocate through Tim 1 1. Further characterization of which preproteins use

which Tim complex is necessary.

The ATP/ADP carrier and the Pi carrier have been shown to utilize the Tim22

complex which was shown to be different from the Tim23-Tim17-Tim44 complex

(Sirrenberg et al., 1996). Although in another study, Tim23 has also been shown to be

needed for the insertion of the ATPIADP carrier (Haucke and Schatz. 1997). It is possible

that these complex proteins which do not contain MTS's, are inserted into the inner

membrane via a different translocation complex, although this is not clear at this point in

time.

5.4 Systems to Determine Tom and Tim Function

In order to son out the involvement of the different Tom and Tim components in the

sorting of preproteins, it is necessary to do the import of the preproteins, containing

different types of sorting signals, in yeast mutants with different components of Tom

and/or Tim knocked out. That is, examine the role of each component of Tom and Tim, in

the translocation of proteins which contain different types of targeting sequences. The

studies which first identified Tim 1 1 and Tim22 and determined their roles in the import of

cytochrome b2 and the ATP/ADP and Pi carriers, respectively, involved these types of

experiments (Sirrenberg et al., 1996; Tokatlidis et al., 1996). As well, the examination of

the targeting pathway of NCBR using ATim 1 I and ATom7 mitochondria has shown that

Tom7 is needed to allow the transfer of proteins from Tom to Tim, and that Tim1 1 is

needed for the arrest of the NCBR hydrophobic sorting signal in the IM (Haucke er al.,

1997). This study illustrates the type of results which can be achieved and further studies

such as this are needed to determine the role of the Tim and Tom components with

precursor proteins which contain different targeting signals.

As well, reconstitution systems are being utilized to examine the roles of some of

the components of the translocation machinery in the absence of the other components.

These systems include soluble reconstitution as well as reconstitution in the presence of

membranes or lipids. The early targeting steps of the chaperones, MSF and Hsp70. and

preproteins with the receptors, Tom70 and TomZO, has been examined in a soluble

reconstitution system (Komiya et al., 1997). This work has confirmed that Hsp70 bound

precursors interact with Tom20 and that MSF bound precursors interact with Tom70 even

in the absence of the OM or of the other Tom components. Work with isolated OM

vesicles or [M vesicles has characterized the roles of Tom or Tim in the absence of one

another. OM vesicles can insert OM proteins but cannot translocate proteins which are

destined to translocate into or across the IM (Mayer el a[., 1993; 1995a). The LM vesicles

have shown that IM proteins can be inserted into the LM dependent on Tim23 but without

the presence of Tim44 and mHsp70 (Haucke and Schatz. 1997). Therefore the roles of

Tom and Tim and the translocation motor (Tim44 and mHsp70) in the matrix are beginning

to be dissected from one another. Further studies such as these will elucidate the

contributions of each system and each component therein.

5.5 Phosphorylation of COX IV

Communication between and within cells is often achieved through signal

transduction pathways. One would expect that communication between the nucleus and the

mitochondrion may involve protein kinases and their target proteins. Although many

different phosphoproteins have been reported in the literature, very few of these have been

positively identified. Therefore, in order to identify novel mitochondria1 membrane

proteins that are potential targets for phosphorylation, I incubated mitochondria and

mitochondria1 membranes with [ Y ~ ~ P I - A T P . The Tx-114 extractable protein was then

subjected to ion-exchange and polyacrylamide gel chromatography after which a

phosphorylated protein of approximately 17,000 Da was purified. The protein was

subjected to trypsin digestion and protein sequencing and it was determined to be COX IV,

an IM protein (Steenam and Shore, 1997b).

Further studies on the phosphorylation of COX IV could be carried out to determine

the site or sites of phosphorylation. As well, which kinase(s) andlor phosphatase(s) are

involved in regulating the COX IV phosphorylation remain to be determined.

The finding that COX IV is phosphorylated by a kinase which is present in the

mitochondrion as well as in isolated membranes shows the potential for the regulation of

oxidative phosphorylation through phosphorylation and signal transduction pathways.

5.6 Future Directions

Our understanding of how mitochondria function to import proteins has advanced

enormously in the past few years. The targeting signals of mitochondria1 preproteins and

their role in insertion and orientation of bitopic membrane proteins are being defined.

Although the exact steps of protein-lipid and protein-protein interactions still have to be

determined. The targeting and insertion of more complex proteins into mitochondria, such

as porin, the ATPIADP carrier, the Pi carrier and the uncoupling protein remain to be

discovered.

The components of Tom and Tim are being identified. Along with what these

components are, how they interact with one another is also being determined through the

use of blue native electrophoresis to identify which receptors are present together in iarge

complexes (Dekker et al., 1996; Dietmeier er al.. 1997). As well. the role of each receptor

in interacting with preproteins and the feature of the preprotein which is involved in binding

is starting to be unraveled. These types of studies which have been done with Tom70,

Tom20 and Tom22 (Schlossmann et nl., 1994; Brix et al., 1997; Schleiff et al., 1997a; b;

Schleiff and Turnbull, 1997), need to be further applied to define the exact nature of the

interactions between receptors and the different precursor proteins. The interactions of

individual receptors with chaperones are also beginning to be determined in soluble

reconstitution systems in the absence of the other receptors (Komiya et al., 1997).

Ultimately, the crystallization of the receptors plus and minus precursor proteins or

chaperones will show the actual structures involved. Also, some light

role of the individual Tom and Tim components, Tim22 has been

is being shed on the

identified as being

involved in the import of the ATP/ADP carrier and the Pi carrier (Sirrenberg er (11.. 1996)

and Tim 1 1 as inserting cytochrome b? and NCBR (Tokatlidis e f nl., 1996; Haucke er d.,

1997). Tom7 has been shown to be necessary for the insertion of porin into the OM

(Honlinger et al., 1996) and for the transfer of proteins from the Tom complex to the Tim

complex (Haucke rr nl., 1997). These recent developments cm help us to understand how

different hydrophobic regions within different contexts in preproteins can be recognized by

the Tom andfor Tim. This is just the beginning of our understanding, further studies with

knock out mutants of the Tom and Tim components along with different mutants of

preproteins targeting signals will define the interactions needed for sorting and insertion of

mitochondria1 proteins. Again, binding studies with isolated components of Tim. similar to

those which have been performed with Tom20, will also be of interest. The role each

machinery plays in the import of precursors is being determined using OM and IM vesicles

(Mayer cr d., 1993: 1995a; Haucke and Schatz. 1997). The identification of the export

machinery involved in conservatively sorted proteins or for proteins synthesized in the

matrix (Hart! and Neupen, 1990) has not yet occurred. It remains to be determinrd

whether separate export machinery does indeed exist. If so, it is predicted to be different

from the bacterial-type Sec machinery (Glick and von Heijne, 1996). The sorting of

proteins from the matrix, i.e., conservatively sorted and mitochondrially synthesized

proteins is just beginning to be examined and requires further work to define the targeting

signals involved (Hermann e t a[., 1995; 1997; Rojo et d.. 1995).

The role of the phosphorylation oECOX IV needs to be elucidated. Does it function

in signalling for respiration4? Is there a function of phosphorylation of preproteins or

components of the machineries in import such as in chloroplasts (Waegemann and Soll,

1996)? Are there other proteins, either phosphorylated or not which interact with

phosphory lated COX IV? Are other phosphoproteins and signal transduction pathways

present in mitochondria and what is their function?

The communication systems between the nucleus and the mitochondrion are

complex and have evolved to include signals within preproteins as well as receptors and

import machinery within the two membranes, the OM and IM. Potential signal

transduction systems may also exist involving kinases, phosphatases and phosphoproteins.

The elucidation of these systems and their interconnections is important not only for

determining mitochondria1 biogenesis, but also for understanding how the whole cell

functions, because of the important role mitochondria play in the cell.

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Original Contributions to Knowledge

The following findings contained within this thesis constitute original contributions to the

field.

1) The net positive charge or length of the hydrophilic region of a signal-anchor are not

important determinants in conferring protein topology in the outer membrane of

mitochondria.

2) The retention of a signal-anchor on the cytosolic side of the outer membmne of

mitochondria correlates with the ability of the hydrophilic region to function independently

as a matrix-targeting signal.

3) The retention of a signal-anchor on the cytosolic side of the outer membrane of mitochondria is due to the amphiphilicity of the hydrophilic region of the signal-anchor.

4) The amphiphilic region of the signal-anchor has lipid-binding capabilities which

contribute to its retention on the cytosolic side of the outer membrane.

5) Not all hydrophobic regions downstream of a matrix-targeting signal will function as

stop-transfer regions and become transmembrane domains in the inner membrane.

6) The insertion of a potential transmembrane segment into the inner membrane requires a relatively high net hydrophobicity when this segment is downstream of a strong matrix-

targeting signal.

7) The inner membrane translocation machinery has a different specificity versus the outer

membrane machinery for the insertion of a protein containing a hydrophobic region

contiguous to a matrix-targeting signal.

8) The targeting of hydrophobic dosains to different compartments within the

mitochondrion depends on several factors: I ) its net hydrophobicity, 2) whether or not it is

permitted to pass across the outer membrane, 3) its distance from a matrix-targeting signal

and 4) the relative strength of the matrix-targeting signal.

9) Cytochrome c oxidase subunit IV can be phosphorylated.

10) Cytochrome c oxidase subunit IV is phosphorylated by a kinase whose activity is

expressed in both whole mitochondria and in isolated mitochondria1 membranes.

APPENDIX:

Construction of Plasmids used in Chapters 2 and 3.

A.1 Constructs-Chapter 2

The construction of the pSP64 expression plasmids encoding pOMD29 and PO-OMD

were as described previously (Li and Shore, 1992a; 1 W2b; McBride et al., 1992). These

plasmids were further manipulated using standard recombinant DNA technology and

polymerase chain reaction (PCR) to create the thirteen other mutants used in this study.

pSP(p0-OMD K3) was created from pSP(p0-OMD) using three PCR reactions.

Codons encoding lysine at positions 1 I and 16 were replaced with those encoding

nsparagine, employing as the upstream primer: S'GCTTGGGCTGCAGGTCGACW

(primer A) and as the downstream primer:

S'ACCATGGAAGTGTGAGCATTTCTAAGAGCTGCATTGTTCAGCAGGATCCT

(23'. The codon encoding lysine at position 29 was replaced with one encoding glutamine,

employing the upstream primer:

S I G C T C A C A C I T C C A T G G T T C G A A A l l l r C G G T A T G G G C A A T

CAAGT3' and the downstream primer: StTTCGAGCTCGCCCGGGGATC3' (primer

B). The PCR products resulting from the above two reactions, which have an overlap of

17bp, were then used as the template DNA with primer A upstream and primer B

downstream. This final PCR product was introduced into PstUSrnd-digested pSP64.

pSP(p0-OMD K3) was further mutated to create pSP(p0-OMD KR4) in which the codon

for arginine at position 26 was converted to encode glutamine. This manipulation

employed as upstream primer:

S'ATGGTTCGAAATTTTCAGTATGGGCAACCAGTCC3' and as downstream

primer, primer B. The resulting PCR product was introduced into BstBYSmai-digested

pSP(p0-OMD K3). pSP(p0-OMD KR4QS) was derived from pSP(p0-OMD KR4).

Three PCR reactions were performed to convert the codons for leucine at positions 5,8,9

and 14 and for isoleucine at position 7 to codons for either serine or glutarnine (positions 8

and 14), employing primer A upstream and as downstream primer:

5'GGAAGTGTGAGCATCTCTGAGCTGCAmGTTCGACTGGCTCCTCGAATT

AGACAGCATCTTCTCTTGCT3'. The codons for methionine at position 2 1, valine at

position 22 and phenylalanine at position 25 were converted to codons for either serine or

glutamine (position 22) using the upstream primer:

5'GAAATGCTCACACTTCCAGTCAGCGAAATTCTCAGTATGGGCMCCAGTC

C3' and downstream, primer B. The PCR products resulting from the above two

reactions, which have an overlap of 17bp. were then used as the template DNA with primer

A upstream and primer B downstream. This final PCR product was introduced into

PstUSmaI-digested pSP64.

A unique restriction site (KpnI), was introduced following the codon for amino acid

10 in pSP(pOMD29), using three PCR reactions. The site was introduced using the

upstream primer: S9AATACAAGCTTGGGCTGC3' (primer C) and as downstream

primer:

S'ACTGCAGCCAAAATGGCGGTACCTGTCTTGTTCCGTAAT3'. In the

second reaction, to amplify the DNA from the codon for amino acid I 1 onwards, the

upstream primer was: 5'GCCATTTTGGCT GCAGT3' (primer D) and downstream

primer B was used. The PCR products resulting from the above two reactions, which

overlapped by 17bp, were then used as template DNA with primer C upstream and primer

B downstream. This final PCR product was introduced into HindIIUSrnaI-digested

pSP64. This construct was designated pSP(pOMD29 k). To create pSP(pOMD29 R) and

pSP(pOMD29 R'), the following two oligos were annealed and introduced into

pSP(pOMD29 k) digested with KpnI.

StCAAGAGCTTCATTACAAGGAACAAGACAGGTAC3' (oligo A) and

5'CTGTCTTGTTCCTTGTAATGAAGCTCTTGGTAC3' (oligo B). Two different

clones were then selected by sequencing, one with the oligos inserted in the forward

orientation pSP(pOMD29 R) and one with the oligos inserted in the reverse orientation

pSP(pOMD29 R').

The plasmids described above were then further mutated to delete the hydrophobic

regions of the encoded proteins. Four different template DNA's, pSP(p0-OMD), pSP(p0-

OMD K3), pSP(p0-OMD KR4) and pSP(p0-OMD KR4QS) were mutated using three

PCR reactions each. In the first reaction, to loop out the coding region for amino acids 38-

58, for d l four templates, the upstream primer was: 5lGATTTAGGTGACACTATAG3'

(primer E) and the downstream primer was:

S1ACGATGCAGTTCAATGGGCCCTGTACTTGACTCTGG3' In the second

reaction, to amplify the DNA coding for DHFR onwards, the primers were, upstream:

S'GGTCCATTGAACTGCAT3' (primer F) and downstream. primer B. For the second

reaction, the template DNA was pSP(pOMD29 A 16-29) (McBride er of., 1992). For each

reaction, the PCR products resulting from the above two reactions, which overlapped by

17bp, were then used as the template DNA with primer E upstream and primer B

downstream. The final four PCR products were inrroduced in to PstUSmaI-digested

pSP64. The resulting plasrnids were designated pSP(p0-OMD A), pSP(p0-OMD K3 A),

pSP(p0-OMD KR4 A) and pSP(p0-OMD KR4QS A).

To delete the transmembrane domain of the proteins coded for from the

pSP(pOMD29) series of constructs, PCR was performed using pSP(pOMD29 A 16-29) as

the template DNA. Three PCR reactions were performed. To delete the codons for amino

acids 11-15 and insert a KpnI site, the upstream primer was primer E and the downstream

primer was:

S'ATGCAGTTCAATGGACCGGTACCTGTCTTGTTCCTTGTAAT3. The

second PCR reaction was the same as the second reaction for the PO-OMD A series. The

PCR products resulting from the above two reactions, which overlapped by 17bp. were

then used as the template DNA with primer E upstream and primer B downstream. The

resulting PCR product was introduced into HindIWSmd-digested pSP64. The resulting

construct pSP(pOMD29 k A) was then digested with KpnI and the oligos A and B were

annealed and inserted. Clones were then selected as outlined above for pSP(pOMD29 R)

and pSP(pOMD29 R'), resulting in pSP(pOMD29 R A) and pSP(pOMD29 R' A).

A.2 Constructs-Chapter 3

As shown above, pSP(pOMD29) and pSP(p0-OMD) also named pSP(p0-SA36) in

this chapter were as previously described. pSP(p0-DHFR) (Skerjanc et dl . , 1990) and

pSP(p0CT) (Nguyen et ol., 1986) have also been described previously.

Two constructs were created based on pSP(pOCT), pSP(p0-SA 14 1) and pSP(p0-SA

242), in which the transmembrane region to the end of pOMD29 was inserted after either.

amino acids 1-141 or after amino acids 1-242 of pOCT, respectively. To construct the

plasmid encoding PO-SA 141, a PCR reaction was performed to amplify the region

encoding the transmembrane region (amino acid 1 I ) to the end of pOMD29. Primer D was

used upstream and downstream, primer B was used with the template DNA of

pSP(pOMD29). The PCR fragment was then inserted into XhoI digested pSP(pOCT),

previously filled in with Klenow. To construct the plasmid encoding PO-SA 242, both

pSP(p0CT) and pSP(pOMD29 k) were digested with KpnI and EcoRI. The resulting

fragment from pSP(pOMD29) was then inserted into the cut vector, pSP(p0CT) resulting

in the DNA encoding the transmembrane region to the end of pOMD29 being placed after

the DNA which encodes amino acids 1-242 in pOCT.

In order to delete the transmembrane regions from the above two constructs, a PCR

reaction employing primer F upstream and primer B downstream on the template

pSP(pOMD29) was performed to amplify the fragment encoding DHFR onwards. This

fragment was then inserted into XhoI digested pSP(pOCT), previously filled in with

Klenow. This plasmid was named pSP(p0-SA 141 A). In order to construct pSP(p0-SA

242 A), three PCR reactions were performed. The DNA encoding the transmembrane

region (amino acids 243-261) was looped out by using pSP(p0-SA 242) as the template

DNA and primer E upstream with:

S'ATGCAGTTCAATGGACCGGTACCATTCTCCTTGGC3' as the downstream

primer. The second reaction amplified the region encoding for DHFR onwards by using

pSP(p0-SA 242) as the template and primer F upstream and primer B downstream. The

resulting DNA from these two reactions overlapped by 17bp and they were then used as the

template DNA for the final reaction with primer E upstream and primer B downstream.

This final PCR product was introduced into pSP64 digested with SmaI and XbaI.

The final construct which was made from pSP(p0-SA 14 1) was pSP(p0-SA 14 1-14).

The DNA was altered such that the alanines of the encoded protein at positions 14. 15, 17

and 18 of the transrnembrane domain (i.e. amino acids 145, 146, 148 and 149) were

mutated to four isoleucinrs. Three PCR reactions were performed, the first introduced the

mutations by using pSP(pOMD29) as the template DNA with the upstream primer:

5'TAGCTCGAGCCATmGATTATAGnATTATTACAGGTACTGCCATCGG3'

and downstream. primer B. The second reaction amplified the DNA encoding the

beginning of the protein until 3 amino acids past the junction with the transmembrane

region. pSP(p0-SA 14 1) was the template with the primer E upstream and the downstream

primer was: S1CAAAATGGCTCGAGCTA3'. These two PCR products which

overlapped each other by 17bp were then used as the template DNA in the final reaction

with primer E upstream and primer B downstream. The resulting DNA was inserted into

Srnal/Xbal digested pSP64.

The authenticity of all of the mutated plasmids was confirmed by nucleotide sequence

analysis.