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Proceedings of the I n t e r n a t i o n a l Conference on the Biogenesis of M i t o c h o n d r i a Bari, Italy, June 2 5 - 2 8 , 1 9 7 3

THE BIOGENESIS OF MITOCHONDRIA T r a n s c r i p t i o n a l , T r a n s l a t i o n a l and Genetic Aspects

edited hy A. M . Kroon Laboratory of Physiological Chemistry State University, G r o n i n g e n , The Netherlands

C. Saccone I n s t i t u t e of B i o l o g i c a l Chemistry University of Bari, Bari, Italy

Academic Press, I n c . 1 9 7 4 A Subsidiary o f H a r c o u r t Brace Jovanovich, Publishers

C O P Y R I G H T © 1974, BY A C A D E M I C PRESS, INC. A L L RIGHTS RESERVED. N O PART O F THIS PUBLICATION M A Y BE REPRODUCED OR T R A N S M I T T E D IN A N Y F O R M OR BY A N Y M E A N S , E L E C T R O N I C OR M E C H A N I C A L , INCLUDING PHOTOCOPY, RECORDING, OR A N Y INFORMATION STORAGE AND RETRIEVAL S Y S T E M , W I T H O U T PERMISSION IN WRITING F R O M T H E PUBLISHER.

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U n i t e d Kingdom E d i t i o n published by A C A D E M I C P R E S S , I N C . ( L O N D O N ) L T D . 24/28 Oval Road. London NW1

Library of Congress Cataloging in Publication Data

International Conference on the Biogenesis of Mitochondria, Rosa Marina, Italy, 1973. The biogenesis of mitochondria.

1. Mitochondria-Congresses. 2. Protein biosyn-thesis-Congresses. I. Kroon, A. M., ed. II. Sac-cone,C.,ed. III. Title. [DNLM: 1. Mitochondria-Metabolism-Congresses. QH603.M5 1601b 1973] QH603.M5I55 1973 574.8'734 73-5314 ISBN 0-12-426750-5

PRINTED IN T H E UNITED STATES OF AMERICA

Contents List of Contributors xi

Preface xxi

Introduction 1

E. Quagliariello

Part I. Mitochondrial Transcription: The Mechanisms and Products, the Role of Mitochondrial DNA and Some Genetic Aspects

Molecular approaches to the dissection of the mitochondrial genome

in HeLa cells 9

G. A t t a r d i , P. Costantino and D . Ojala

The DNA-dependent RNA polymerases from yeast mitochondria 31

T. R . Eccleshall and R . S. Criddle

A mitochondrial DNA-directed RNA Polymerase from yeast

mitochondria 47

A . H. Scragg

The DNA-dependent RNA Polymerase from rat-liver mitochondria 59

R . Gallerani and C. Saccone

Expression of the mitochondrial genome in wild type and in an

extranuclear mutant of Neurospora crassa 71

H. Küntzel, I. A l i and C. Blossey

Transcription of mtDNA by mitochondrial RNA Polymerase from

Xenopus laevis 79

/. B . D a w i d and G-J. Wu

v

CONTENTS

Effect of rifampicin on macromolecular synthesis in rat-liver

mitochondria 85

M. N G a d a l e t a and C. Saccone

Characterization of yeast grande and petite mitochondrial DNA by

hybridization and physical techniques 89

M. Rabinowitz, J. Casey, P. G o r d o n , J. Locker, H-J. Hsu and G. S. Getz

Elongation factors for organellar protein synthesis in Chlorella vulgaris 107

O. Ciferri, O. T i b o n i , G. L a z a r and J. van E t t e n

Synthesis and processing of mitochondrial ribosomal RNA in wild

type and poky strain of Neurospora crassa 117

Y. K u r i y a m a and D . J. L . L u c k

Biogenesis of mitochondria from A r t e m i a salina cysts and the

transcription in vitro of the DNA 135

H. Schmitt, H. Grossfeld, J. S. Beckmann and U. Z . L i t t a u e r

Structure of petite mtDNA; prospects for mitochondrial gene

isolation, mapping and sequencing 147

P. Borst

The purification of mitochondrial genes using petite mutants

of yeast 157

P. Nagley, E . B . G i n g o l d and A . W. L i n n a n e

Interaction of mitochondrial protein synthesis on the regulation

of gene activity in Saccharomyces cerevisiae 169

P . P. Puglisi and A . A . A l g e r i

Isolation, purification, physico-chemical characterization and

recombination of yeast mitochondrial DNA Segments conferring

resistance either to chloramphenicol or to erythromycin 177

H. F u k u h a r a , J. Lazowska, F. M i c h e l , G F a y e , G. M i c h a e l i s , E. Petrochilo and P. Slonimski

Mitochondrial genetic factors in the cellular response to

chlorimipramine in Saccharomyces cerevisiae 179

D . Linstead, I. Evans and D . Wilkie

Mitochondrial genetics 193

A . W. L i n n a n e , N . H o w e l l and H. B. L u k i n s

v i

CONTENTS

Mitochondrial genes and ATP-synthetase 215

D . E. Griffiths, R . L . H o u g h t o n and W. E . L a n c a s h i r e

Characterization of a respiration competent, oligomycin resistant

mutant of Schizosaccharomyces pombe lacking cytochrome

6-566 but still binding antimycin 225

W. Bandlow

Mitochondrial enzymes in man-mouse hybrid cells 231

V. van H e y n i n g e n , J. W. Craig and W. Bodmer

Temperature-dependent changes in levels of multiple-length

mitochondrial DNA in cells transformed by a thermosensitive

mutant of Rous Sarcoma Virus 245

M. M. K. Nass

Propagation and recombination of parental mtDNAs in

hybrid cells 255

/. B . D a w i d , I. H o r a k and H. G. Coon

Mitochondrial mutations in Paramecium: phenotypical

characterization and recombination 263

A . Adoutte

Part II. Characteristics of the Mitochondrial Protein Synthetic Machinery

Mutations affecting mitochondrial ribosomes in yeast 275

L . A . G r i v e l l

Evidence of involvement of mitochondrial polysomes and

messinger RNA in synthesis of organelle proteins 289

C. S. Cooper and C. J. Avers

Mitochondrial polysomes from Neurospora crassa 305

E. Agsteribbe, R . D a t e m a and A . M . K r o o n

Nascent Polypeptide chains on mitochondrial ribosomes and

their integration into the inner mitochondrial membrane 315

R . M i c h e l and W. Neupert

Analysis of mitoribosomes from T e t r a h y m e n a by Polyacrylamide gel electrophoresis and electron microscopy 327

B . J. Stevens, JrJ. Curgy, G. Ledoigt and J. A n d r e

vn

CONTENTS

Fine structure of mitochondrial ribosomes of locust flight muscle 337

W. Kleinow, W. Neupert and F. Miller

The structure, composition and function of 55S mitochondrial

ribosomes 347

T. W. O'Brien, D . N . Denslow and G. R . Martin

Physicochemical and functional characterization of the 55S

ribosomes from rat-liver mitochondria 357

H. de Vries and A . M. Kroon

Characterization of the monomer form of rat-liver mitochondrial

ribosomes and its activity in poly U-directed polyphenylalanine

synthesis 367

M. Greco, G. Pepe and C. Saccone

RNA from mitochondrial ribosomes of Locusta m i g r a t o r i a 377

W. Kleinow

Coding degeneracy in mitochondria 383

N. C h i u , A . O. S. Chiu and Y. Suyama

On the sensitivity of mammalian mitochondrial protein synthesis

to inhibition by the macrolide antibiotics 395

A . M. K r o o n , A . J. Arendzen and H. de Vries

Part III. Synthesis of Mitochondrial Proteins

Mitochondrial products of yeast ATPase and cytochrome oxidase 405

A . Tzagoloff, A . A k a i and M. S. R u b i n

Initiation, identification and Integration of mitochondrial proteins 423

H. R . Mahler, F. F e l d m a n , S. H. P h a n , P. H a m i l l and K. Dawidowicz

The biosynthesis of mitochondrial ribosomes in Saccharomyces cerevisiae 443

G. S. P. Groot

Cooperation of mitochondrial and cytoplasmic protein synthesis

in the formation of cytochrome c oxidase 453

W. Sebald, W. M a c h l e i d t and J. Otto

Studies on the control of mitochondrial protein synthesis

in yeast 465

D . S.Beattie, L - F . H. L i n and R . N. Stucheil

v n i

CONTENTS

The biosynthesis of mitochondrial cytochromes 477

E. Ross, E . Ebner, R . O. Poyton, T. L . M a s o n , B. Ono and G. Schatz

Biogenesis of cytochrome b in Neurospora crassa 491

H. Weiss and B . Z i g a n k e

Precursor proteins of cytochrome c oxidase in cytochrome c oxidase deficient copper-depleted Neurospora 501

A . J. Schwab

Antibodies to subunits of cytochrome c oxidase and their relation to precursor proteins of this enzyme 505

S. Werner

Cytoplasmic ribosomes associated with yeast mitochondria 511

R . E . Kellems, V. F. Allison and R . W. Butow

Mitochondrial synthesis of glycoproteins and surface properties

of mitochondrial membranes 525

H. B . Bosmann and M. W. Myers

Membrane structure and mitochondrial biogenesis 537

L . Packer and L . W o r t h i n g t o n

Lecithin formation in phospholipase otreated rat-liver

mitochondria 541

B . C. van Schijndel, A . Reitsema and G. L . Scherphof

Concluding remarks 545

C. Saccone and A . M. Kroon

ix

NASCENT POLYPEPTIDE CHAINS ON MITOCHONDRIAL RIBOSOMES AND THEIR INTEGRATION INTO THE INNER MITOCHONDRIAL MEMBRANE

R. Michel and W. Neupert

I n s t i t u t für P h y s i o l o g i s c h e C h e m i e u n d P h y s i k a l i s c h e

B i o c h e m i e d e r Universität, 8 0 0 0 München 23 G o e t h e

s t r a s s e 3 3 , F e d e r a l Repüblic o f Germany.

L i t t l e i s known about the mechanism how mitochon

d r i a l t r a n s l a t i o n products are transferred from t h e i r

s i t e of synthesis to t h e i r s i t e of function, i . e . from

the mitochondrial ribosomes into enzyme complexes of

the inner mitochondrial membrane (1-4). The pe c u l i a r

nature of the mitochondrial t r a n s l a t i o n products may

play a d i s t i n c t r o l e i n th i s process. Therefore we ha-

ve studied some properties of mitochondrial t r a n s l a

t i o n products before and a f t e r t h e i r i n t e g r a t i o n into

the membrane.

RESULTS.

P r o p e r t i e s o f n a s c e n t t r a n s l a t i o n p r o d u c t s o n m i t o

c h o n d r i a l r i b o s o m e s . After l a b e l l i n g whole N e u r o s p o

r a c e l l s with radioactive amino acids i n the presence

of cycloheximide (CHI), i t i s possible to i s o l a t e mi

tochondrial ribosomes i n which the nascent peptide

chains are r a d i o a c t i v e l y l a b e l l e d , whereas the riboso-

mal proteins are not. This i s due to the f a c t , that

the proteins of mitochondrial ribosomes are formed at

the CHI s e n s i t i v e cytoplasmic ribosomes (5-7). By this

way, we have an experimental System i n which the nas

cent t r a n s l a t i o n products of mitochondria can e a s i l y

be i d e n t i f i e d and analysed.

A) Mitochondrial ribosomes carrying nascent peptide

chains.

F i g . 1 shows the r e s u l t of density gradient c e n t r i -

fugation of mitochondrial ribosomes from c e l l s exposed

315

Fig. 1. Sucrose density gradient centrifugation of mitochondrial ribosomes with radioactively labeled nascent peptide chains. A: Optical density pattern (260nm); B: Radioactivity pattern; C: Specific radioactivity related to absorbance at 260 nm. Cells were incubated with cycloheximide (100 ug/ml) for 2.5 min, [

JH]leucine

was added (1 uCi/ml) and after 1.5 min the cells were rapidly cooled to 0° C. Mitochondrial ribosomes were isolated and subjected to gradient centrifugation (9). Fraction 1 is top, and fraction 60 bottom of the gradient.

Fig. 2. Effect of incubation of mitochondrial ribosomes in AMT buffer at 37° C on the distribution of radioactivity in the sucrose density gradient. Mitochondrial ribosomes were labeled as described in Fig. 1. They were kept at 37° C in AMT buffer (0.1 M N H 4 C I , 10 mM MgCl2, 10 mM Tris-HCl, pH 7.5) for the time pe-riods indicated in the figure. In the case of the radioactivity associated with the wall of the centrifuge tube the radioactivity in the control sample (8250 counts/min) was subtracted from a l l values.

316

NASCENT POLYPEPTIDE CHAINS ON MITOCHONDRIAL RIBOSOMES

to a pulse of radioactive leucine i n the presence of

CHI, In the o p t i c a l density pattern ( F i g . 1A) the peak

of the monomeric ribosomes i s prominent. A considera-

b l e amount of polymeric ribosomes (ca. 45 % of t o t a l

ribosomes) i s present. In the l a b e l l i n g pattern ( F i g .

1B) monomeric, dimeric and higher polymeric ribosomes

also can be distinguished. However, the majority of

the r a d i o a c t i v i t y (ca. 85 %) i s found associated with

polymeric ribosomes. Accordingly, the s p e c i f i c r a d i o

a c t i v i t y , related to A260nm ( F i g . IC) i s about f i v e

times higher i n the polymeric ribosomes. This d i f f e r -

ence i n s p e c i f i c r a d i o a c t i v i t y of monomeric and poly

meric ribosomes suggests, that monomers are not mere-

l y breakdown products of polymers. This i s i n contrast

to cytoplasmic ribosomes, where monomers and polymers

have the same s p e c i f i c r a d i o a c t i v i t y a f t e r short p u l

se l a b e l l i n g .

The amount of t o t a l r a d i o a c t i v i t y i n the polymer

region i s subject to large a l t e r a t i o n s which depend

on the preparation conditions of the ribosomes. How

ever, the less the amount of r a d i o a c t i v i t y i n the po

lymer region i s , the more r a d i o a c t i v i t y appears i n the

p e l l e t of the gradient. The y i e l d of r a d i o a c t i v i t y at

the monomer i s rather constant. This leads us to sup-

pose that the polymeric ribosomes have a high tenden-

cy to aggregate.

In order to test t h i s , mitochondrial ribosomes we

re kept i n Mg containing buffer at 37° C for d i f f e r -

ent time periods and then subjected to gradient cen-

t r i f u g a t i o n . In F i g . 2 the r a d i o a c t i v i t y found i n the

d i f f e r e n t f r a c t i o n s of the gradient i s shown. Düring

the time period of incubation the r a d i o a c t i v i t y asso

ciated with the monomer remains constant a f t e r a

s l i g h t i n i t i a l decrease. In contrast, the r a d i o a c t i

v i t y at the dimer and higher polymers decreases

strongly. The r a d i o a c t i v i t y disappearing from this r e

gion does not appear at the top of the gradient, but

can be traced i n the p e l l e t and at the wall of the

centrifuge tube.

This s e l e c t i v e tendency of polymeric ribosomes to

aggregate and form heavy p a r t i c l e s gives r i s e to the

317

A B

J V° \ „/> 1 Ii

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500 fc

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2000 1

Fig. 3. Treatment of mitochondrial ribosomes with ribonuclease. Ribosomal sus-pensions in AMT buffer were kept at 0° C for 60 min with and without pancreatic ribonuclease, and then subjected to gradient centrifugation. o—o : radioactivity; : absorbar.ce at 260 nm. A, control (without ribonuclease); B, 1 jg/ml ribonuclease; C, 4 ug/ml ribonuclease; D, 20 ug/ml ribonuclease.

10 20 x 40 so so Numb« of g«l shce

Fig. 4. Gel electrophoretic analysis cf radioactively labeled nascent peptide chains associated with mitochondrial polymeric ribosomes. A, Ribosomes dissolved in 0.1 M Tris-HCl, 0.5 % SDS, pH 8, and kept for 1 h at 37° C; B, Ribosomes dis solved as in A, plus 20 ug/ml pancreatic ribonuclease.

318


question whether the polymeric ribosomes are r e a l mes-

senger-ribosome complexes or aggregates themselves.

Treatment with ribonuclease i s one way to test t h i s .

Cytoplasmic polymeric ribosomes are already converted

to monomers to a considerable extent by incubation

with 0.5 ug/ml ribonuclease f o r 60 min at 0° C. This

conversion i s c o m p l e t e at a concentration of 20 yg/ml

ribonuclease. In contrast, the o p t i c a l density pattem

of mitochondrial ribosomes i s not changed by incuba

t i o n with corresponding ribonuclease concentrations

( F i g . 3). Also no change i s observed i n the r a d i o a c t i

v i t y pattern. This suggests that mitochondrial poly

meric ribosomes are aggregates. In agreement with this

i s the O b s e r v a t i o n t h a t a f t e r t r e a t m e n t of i s o l a t e d

mitochondria with puromycin, aggregation of ribosomes

is almost completely a b o l i s h e d , Therefore i t appears

that nascent chains are responsible for th i s aggrega

t i o n . Similar observations and conclusions were made

by Ojala and A t t a r d i (8).

B) Properties of nascent t r a n s l a t i o n products.

On the basis of these findings we are led to con-

clude that the peptide chains on mitochondrial r i b o

somes have c e r t a i n p e c u l i a r p r o p e r t i e s . This i s under-

l i n e d by the Observation that these peptides i n the

form of peptidyl-transfer-RNA cannot be separated from

the ribosomal proteins by treatment with phenol, un-

less SDS i s present. If SDS i s removed from SDS solu-

b i l i z e d ribosomes, the nascent peptides become inso-

l u b l e .

In order to further elucidate these p r o p e r t i e s , i t

was examined whether the nascent peptide chains can

be removed from the mitochondrial ribosomes as pepti-

dyl-puromycin and as peptidyl-tRNA. To control the ex-

perimental setup, f i r s t cytoplasmic ribosomes were

tested. In this case, treatment of ribosomes with pu

romycin, GTP and G-factor leads to the release of the

nascent chains i n a soluble form. A l s o , upon exposure

of cytoplasmic ribosomes to EDTA, d i s s o c i a t i o n into

subunits and release of peptidyl tRNA occurs. In con

t r a s t , when mitochondrial ribosomes are treatedwith

puromycin, GTP and G-factor, the radioactive nascent

319

400

300 j

200 J

100 <

0 .

6 0 0 -

5 0 0 -

400

300

200

100

0

30C

200

100

0

A / 1 i 1 i 1

1 1 \ t I

1 1 B

i

1 i i

4

1

1 1 /

* t 0

C

1 C \

f 1

1

J 1 ,

1 VW — i 1

1 10 20 30 40 50 60 Number of gel slice

Fig. 5. Gel electrophoretic anaiysis of radioactively labeled nascent peptide chains associated with mitochondrial monomeric ribosomes. A, Ribosanes pretreated as described for Fig. 4A; B, Ribosomes dissolved in 0.1 M phosphate buffer, pH 11, and kept for 1 h at 37° C; C, Ribosomes pretreated as described for Fig. 4B.

320


chains disappear from monomers and p o l y m e r s i n the

gradient, however t h e y are not released i n a soluble

form, but are found at the wall and i n the p e l l e t of

the tube a f t e r c e n t r i f u g a t i o n . Treatment with EDTA

disso c i a t e s the mitochondrial ribosomes but the radio-

active chains remain associated with the large subu-

n i t s and with r i b o s o m a l structures which c a n n o t be

c l e a r l y i d e n t i f i e d on the b a s i s of t h e i r S e d i m e n t a t i o n

behaviour. They probably represent aggregates of the

large subunit (9). I t appears from these observations

that the nascent chains on mitochondrial ribosomes are

not watersoluble but aggregate i n aqueous Solutions.

For further anaiysis of the nascent chains the r i

bosomes were dissolved i n SDS containing buffer and

subjected to gel electrophoresis i n the presence of

SDS. This was done separately for poly- and monomeric

ribosomes. When nascent chains on cytoplasmic r i b o s o

mes were studied they showed a scattered d i s t r i b u t i o n

of apparent molecular weights (AMWs). No s i g n i f i c a n t

d i f f e r e n c e between chains of monomeric and polymeric

ribosomes i s seen (9). In F i g . 4A gel electrophoresis

of r a d i o a c t i v e l y l a b e l l e d chains on mitochondrial po

lymeric ribosomes i s shown. The AMWs also appear to

be quite spread here. F i g . 4B represents the d i s t r i

bution a f t e r treatment of the SDS s o l u b i l i z e d polyme

r i c ribosomes with ribonuclease. This was done to de-

grade p e p t i d y l tRNA which might s t i l l be present a f

ter incubation of the ribosomes at pH 8 and 37° C f o r

one hour. The minor changes compared to F i g . 4A and

the Observation that tRNA on this gel migrates corres-

ponding to an AMW of ca. 15,000 suggest that hydroly-

s i s of the peptide-tRNA bond has already occurred to

a large extent.

In F i g . 5A an e l e c t r ö p h o r e t i c S e p a r a t i o n of c h a i n s

associated with mitochondrial monomeric ribosomes i s

presented. A peak with an AMW of 27,000 i s prominent

i n a ddition to a double peak with an AMW i n the ränge

of 8,000 - 12,000. I f the ribosomes are immediately

subjected to gel electrophoresis a f t e r having been

d i s s o l v e d , mainly the f i r s t peak i s present. Prolong-

ed incubation of the ribosomes r e s u l t s i n a decrease

321

R. MICHEL AND W. NEUPERT

of the f i r s t peak and i n an increase of the second

one. At pH 6 this process i s slower than at pH 8, and

at pH 11 ( F i g . 5B) i t i s f a s t e r . Treatment of SDS so-

l u b i l i z e d ribosomes with ribonuclease causes the d i s -

appearance of the 27,000 AMW peak. At the same time

the double peak with the lower AMW increases ( F i g . 5C).

Since determinations of AMWs are d i f f i c u l t or even

impossible i n the low molecular weight ränge by gel

el e c t r o p h o r e s i s , gel chromatography was ca r r i e d out.

The chains on the monomeric ribosomes are eluted from

the column (Sephadex G 200) together with the marker

cytochrome e. Af t e r digestion with t r y p s i n smaller

products appear. These are eluted together with the

marker leucine. The chains at the polymeric ribosomes

show a very s i m i l a r behaviour.

These result s suggest that the 27,000 AMW peak re-

presents peptidyl tRNA and that the peptides have AMWs

of 8,000 - 12,000. This i s substantiated by the O b s e r

vation already mentioned that transfer RNA migrates

with an AMW of about 15,000.

On the basis of the r e s u l t s obtained by gel chro

matography, the AMW of the polymer product i s i n the

same ränge as that of the monomer product. This can

only be reconciled with the electrophoretic data, i f

we assume that on the gel the polymer chains aggrega

te e i t h e r with each other or with the hydrophobic gel

m a t e r i a l . This again would be i n accordance with the

se l e c t i v e tendency of polymeric ribosomes to aggrega

t e .

T r a n s l a t i o n p r o d u c t s a f t e r i n t e g r a t i o n i n t o t h e

membrane. What do the t r a n s l a t i o n products on the

monomeric ribosomes which display a rather uniform

AMW represent ? I f they are completed t r a n s l a t i o n p r o

ducts, then i t should be possible to f i n d them i n the

mitochondrial membrane. To follow this question, Neu

r o s p o r a c e l l s were l a b e l l e d under the following con-

d i t i o n s : CHI was added to the c u l t u r e , a f t e r 2%5 min

radioactive leucine and a f t e r further 2 min a chase

of unlabelled leucine was given. Af t e r 45 min the

c e l l s were cooled, mitochondrial membranes were pre-

pared and subjected to gel electrophoresis. The d i s -

322

Fraction no. Number of gel shce

F i g . 6. Gel electrophoretic anaiysis of mitochondrial membranes after pulse label-

ing with radioactive leucine in v i v o in the presence of cycloheximide. After 2.5

min preincubation of t t e u r o s p o r a c e l l s with cycloheximide, a 2 min pulse of [-*H]-

leucine was given, followed by a chase of unlabeled leucine. In (A) the c e l l s were

immediately cooled after having given the chase, in (B) the chase lasted for 45 min

at growth temperature (25° C).

Fig . 7. Gel electrophoretic anaiysis of mitochondrial membranes after pulse label-

ing with [-̂ H] leucine in v i v o in the presence of cycloheximide followed by a 1 h chase at different temperatures. The temperature during chase is given in the figure.

323


t r i b u t i o n of r a d i o a c t i v i t y on the gel i s shown i n F i g .

6B, Peaks are prominant at AMWs of 36,000, 27,000 and

18,000, and a small one at about 12,000. I f a l l these

protein bands were o r i g i n a l t r a n s l a t i o n products, then

we would have to conclude that we do not recover com-

plet e d t r a n s l a t i o n products on the monomeric ribosomes.

However, i f we stop the l a b e l l i n g procedure immedia-

tely a f t e r having added the chase, a quite d i f f e r e n t

l a b e l l i n g pattern of the membrane i s observed ( F i g .

6A). A large part of the r a d i o a c t i v i t y co-migrates

with cytochrome a or i s even f a s t e r . The high mole-

cular weight peaks are present only to a low extent,

the 18,000 AMW peak to a s i m i l a r extent as a f t e r a

long chase. The background i n the high molecular

weight region i s very high and i t looks l i k e low mo

lec u l a r weight material i s t a i l i n g . The t o t a l radioac

t i v i t y i n the membrane does not change, so i t mustbe

conluded that a conversion of t r a n s l a t i o n products

with lower AMWs to such with higher AMWs takes place

in the membrane. I f chase times i n between those shown

i n F i g . 6 are in v e s t i g a t e d , a gradual s h i f t of the

r a d i o a c t i v i t y i s observed.

This process was studied i n a further experiment.

L a b e l l i n g was performed here as described f o r . F i g . 6,

with the modification that a f t e r giving the chase,

the culture was divided into four equal portions and

these were rapidly adjusted to 0, 12, 22 and 37° C,

res p e c t i v e l y . Incubation at these temperatures was

carri e d on for one hour. F i g . 7 shows the l a b e l l i n g

patterns of the mitochondrial membrane. A chase per

formed at 0° C does not change the l a b e l l i n g pattern

of the unchased c e l l s ( c f . F i g . 6A). At higher tempe

ratures the conversion takes place. It i s the f a s t e r

the higher the temperature i s .

DISCUSSION.

1. The rather uniform apparent molecular weight of

the nascent chains at the mitochondrial monomeric r i

bosomes may have two possible reasons: a) It i s an ar-

t i f a c t , because nascent chains are cut down to a cer-

t a i n length; b) the monomers carry completed chains.

324


P o s s i b i l i t y a) does not appear to be l i k e l y , since no

i n d i c a t i o n for such a breakdown was found under the

d i f f e r e n t conditions of i s o l a t i o n and Separation of

mitochondria and ribosomes. Furthermore, the observed

conversion of t r a n s l a t i o n products i n the membrane

could explain that no larger peptide chains are found

at the mitochondrial ribosomes.

The presence of completed Polypeptide chains at

the monomeric ribosomes i s related to the problern, how

mitochondrial t r a n s l a t i o n products reach the inner

membrane. They are obviously so hydrophobic that i t

seems highly improbable that they are released from

the ribosomes into the matrix i n a soluble form. So

we can speculate that the ribosome leaving the messerr

ger-RNA does not immediately release the product and

d i s s o c i a t e , but rather dissociates only a f t e r having

transported the completed chain to the membrane. For

discussion of the a l t e r n a t i v e mechanism that r i b o s o

mes are bound to the inner membrane, see r e f . 9.

2. Experiments presented here suggest that the mi

tochondrial t r a n s l a t i o n products found i n enzyme com-

plexes such as cytochrome a a ^ 9 cytochrome b9 and ATP

ase (10-13) are not o r i g i n a l t r a n s l a t i o n products, but

are rather generated by conversion of peptides with

lower apparent molecular weights. A f t e r pulse l a b e l

l i n g , the mitochondrially synthesized subunits of cy

tochrome a oxidase appear i n the enzyme protein with

a time course s i m i l a r to that described f o r the

36,000, 27,000 and 18,000 AMW peaks i n the membrane

(14). It i s possible that for each subunit this con

version process i s the rate l i m i t i n g Step.

3. It i s concluded i n this report that mitochondrial

t r a n s l a t i o n products exhibit a strong hydrophobic cha-r a c t e r . This i s substantiated by amino acid analyses

of subunits of cytochrome c oxidase formed inside the

mitochondria (15). On the basis of these data we are

led to suggest that i n mitochondria a System of trans-

c r i p t i o n and t r a n s l a t i o n had to be maintained ( i f we

follow the endosymbiont theory) or had to be created

( i f we follow some other theory (l6)> because the

t r a n s l a t i o n products are so hydrophobic that they can-

325


not be transported through the cytoplasm and the i n -

t e r c r i s t a e space. They rather have to be delivered to

the inner membrane d i r e c t l y , i . e . from the matrix s i -

de.

ACKNOWLEDGEMENT.

This work was supported by the Deutsche Forschungs

gemeinschaft, Schwerpunktprogramm "Biochemie der Mor

phogenese11.

REFERENCES.

1. G. Schatz, i n : "Membranes of Mitochondria and

Chloroplasts", E. Racker Ed., Nostrand Reinhold

Co., New York, p. 251 (1970).

2. H. K ü n t z e l , Curr.Top.Microbiol.Immunol. 54̂ , 94

(1971) .

3. D.S. Be a t t i e , Subcell.Biochem. ±9 1 (1971).

4. P. Borst, Ann.Rev.Biochem. 4j_, 333 (1972).

5. H. K ü n t z e l , Nature (Lond.) 222, 142 (1969).

6. W. Neupert, W. Sebald, A. Schwab, A. P f a l l e r &

Th. Bücher, Eur.J.Biochem. _HD, 589 (1969).

7. P.M. L i z a r d i & D.J.L. Luck, J . C e l l B i o l . 54, 56

(1972) .

8. D. Ojala & G. A t t a r d i , J.Mol.Biol. 65^, 273 (1972).

9. R. Michel & W. Neupert, Eur.J.Biochem. i n press

(1973) .

10. H. Weiss, W. Sebald & Th. Bücher, Eur.J.Biochem.

22,, 19 (1971).

11. T.L. Mason & G. Schatz, J.Biol.Chem. 248, 1355

(1973).

12. H. Weiss, Eur.J.Biochem. 30, 469 (1972).

13. A. Tzagoloff, M.S. Rubin & M.F. S i e r r a , Biochim.

Biophys.Acta 301, 71 (1973).

14. A.J. Schwab, W. Sebald & H. Weiss, Eur.J.Biochem.

30, 511 (1972).

15. W. Sebald, W. Machleidt & A. Otto, Eur.J.Biochem.

i n press (1973).

16. R.A. Raff&H.R. Mahler, Science 177, 575 (1972).

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