7 - mitochondrial protein import in plants

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Plant Molecular Biology 0: 311–338, 1998.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.311

Mitochondrial protein import in plants

Signals, Sorting, Targeting, Processing and Regulation

Elzbieta Glaser1, Sara Sjöling1, Marcel Tanudji2 and James Whelan2

1 Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stock-holm, Sweden; 2 Department of Biochemistry, University of Western Australia, Nedlands 6907, Perth, Western

Australia, Australia

Key words: mitochondrial processing peptidase, molecular chaperones, plant mitochondria, protein import,

processing peptidase, protein processing, protein sorting, regulation of protein import, signal peptides

Abstract

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes andspecific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are

synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which

is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components

and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties

of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition,

mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molec-

ular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial

processing peptidase (MPP), which in contrast to other sources, is integrated into the bc 1 complex of the respi-

ratory chain. This is the most studied component of the plant import machinery characterised to date. What are

the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are

several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage,

specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into

mitochondria.

Introduction

Plant cell constitutes an interesting biogenetic system

in which genetic information is located in three in-

tracellular compartments, the nucleus, mitochondria

and chloroplasts. Despite the fact that both mito-

chondria and chloroplasts contain their own genetic

information, most of the protein complement of these

organelles is synthesised in the cytosol. This results

in an active protein trafficking from the cytosol tothese organelles. Mitochondrial protein import process

has been studied very extensively during the past two

decades, especially in lower eukaryotes, in fungi, and

resulted in characterisation of many signal peptides,

protein translocating machineries on the outer and in-

ner membrane and involvement of both cytosolic and

organeller chaperones in the process of transport (for

reviews see [141, 167]). Although less information

is available on the mitochondrial protein import in

plants (for recent reviews [178, 211]), several new

and unique findings have been reported in the last few

years.

The mitochondrial protein import process is a

multi-step process including the following events:

(1) Synthesis in cytosol of the precursor protein con-

taining an N-terminal extension called a presequence

that functions as an organellar sorting and target-ing signal. (2) Interaction of the newly synthesised

precursor with cytosolic chaperones and other fac-

tors, to confer an import competent conformation

on the precursor, facilitate recognition and prevent

organellar mis-sorting. (3) Recognition of the pre-

cursor on the organellar membrane either by direct

interaction of the presequence with organellar recep-



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tors or by interaction of the presequence bound to

cytosolic chaperones. (4) Translocation of the precur-

sor through the organellar membranes. (5) Proteolytic

processing of the precursor inside mitochondria by

a highly specific mitochondrial processing peptidase.

(6) Assembly of the mature form of the protein into

a functional, oligomeric protein complex in a processinvolving organellar chaperones.

In this chapter we will review our present knowl-

edge on components and mechanisms involved in

mitochondrial protein import in plants with emphasis

on findings that are specific and unique for plants.

Synthesis of precursor proteins in the cytosol

The nuclear encoded mitochondrial proteins carry tar-

geting information as signal peptide located at N-

terminus. Most of the signals constitute an N-terminal

extension, the so called presequence that is cleavedoff by the mitochondrial processing peptidase (MPP)

during, or after, import into the mitochondrion, re-

sulting in the production of mature protein. Only the

outer membrane proteins and some low molecular

mass proteins of the inner membrane contain non-

cleavable signal peptides [169, 211]. We will review

the major features of the plant mitochondrial prese-

quences that lead to sorting, targeting, translocation

and processing the nuclear encoded precursor proteins

into mitochondria.

Presequence composition and structure

A collection of all available plant presequences of

nuclear encoded mitochondrial proteins from the data-

bases shown in Figure 1 contains at present over

100 sequences. Characteristic features of the prese-

quences were outlined using statistical analysis, se-

quence alignment and secondary structure predictions.

Out of a total of 737 mitochondrial precursor se-

quences from different sources, we have analysed

80 plant presequences statistically in terms of length,

amino acid composition and conserved domains [169,

178, 211]. Seventy-one sequences were predicted, or

estimated, to contain a mitochondrial processing pep-tidase (MPP) cleavage site. Out of these, 31 sequences

contained a confirmed/microsequenced cleavage site;

a final collection of 25 different presequences was

used for analysis, i.e. only one sequence representing

each protein was analysed.

The length of the known plant presequences varies

from 13 amino acid residues for the alternative oxidase

of Arabidopsis thaliana to 85 amino acid residues of

the P subunit of glycine decarboxylase from pea, with

an average length calculated to 40 amino acid residues.

Compared to mammalian, yeast and N. crassa prese-

quences, the plant presequences are in average 7–9

residues longer. The plant presequences are rich in

serine (17.1%), arginine (12,6%), alanine (12.0%),leucine (10,6%), but low in cysteine (1.0%), histidine

(1.3%), tryptophane (1.4%), tyrosine (1.4%), glutamic

acid (1.4%) and aspartic acid (1.5%). The amino acid

composition of the plant presequences is thus simi-

lar to that of other mitochondrial presequences [169,

201] i.e. they exhibit high content of basic hydroxy-

lated residues (with the exception for histidine) and

low content of acidic and aromatic residues. A unique

feature is that the plant presequences have higher con-

tent of serine (17%) as compared to that of yeast (7%),

mammals (3%) or N. crassa (10%) [178].

The existence of amino acid sequences for full-

length precursor proteins corresponding to the same

protein from different plant species enables sequence

comparison of both the presequences and mature parts

of the protein. The identity of amino acids of the

mature parts of the precursors was much higher than

within the presequences indicating a common evolu-

tionary pathway previous to the endosymbiotic event.

The acquisition of the presequence, after gene transfer

from the mitochondria to the nucleus, could have been

an independent step for different ancestors of each

species.

In order to investigate if there are any conserved

domains within the plant presequence we aligned pre-sequences from different plant species, corresponding

to the same protein [178]. Proteins for which at least

three sequences are available from different sources,

e.g. F1β, FeS, HSP 60, and superoxide dismutase

(SOD) (with an exception for alternative oxidase) have

higher identity of amino acids in the amino- and

carboxy- terminal regions, than in the central parts

of the presequences. The more conserved N- and C-

terminal regions of these presequences suggest func-

tional importance of these domains. The N-terminal

domain, or the import domain, is important for guid-

ing the precursor protein to the mitochondria. This

domain shows a rather regular alternation between ba-

sic and hydrophobic residues and has the potential to

form amphiphilic α-helix with one positively charged

and one apolar face, in contact with lipids, or other

proteins [200]. The C-terminal domain, or the process-

ing domain, most often contains a cleavage motif for

MPP and a secondary structure which is compatible



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Figure 1. Amino acid sequence of the presequences of the nuclear encoded mitochondrial proteins. The amino acid sequence of the presequence

and the first three amino acids of the mature protein are shown. The space indicates the beginning of the mature protein, if determined

directly by N-terminal sequencing the protein is indicated by an ∗. The N-terminal region of the other proteins was as indicated in the

published sequences (see Ref for details), this was judged on homology with published sequences (from plants and other organisms) for

which N-terminal information was available. 1 indicates proteins that have no cleavable presequence and the first 30 amino acids of the

mature protein are shown or the amino acids that the original authors suggested that were involved in targeting. 2 represents a naturally

occurring plant presequence that targets to the mitochondrion and chloroplast (see text for details). + = Personal communication Michael

Hodges. Abbreviations used: Plant species - At = Arabidopsisthaliana, Bn = Brassica napus, Cr = Chlamydomonas reinhardtii,

Cr u = Chenopodium rubum, Cs = Cucurbita sp, Cv = Citrullus vulgaris, Eg = Euglena gracilis, Eug = Eucalyptus gunnii,

Fp = Flaveria pringlei, Gm = Glycine max, Gv = Gracilaria verrucosa, H b = Hevea Brasiliensis, I b = Ipomea batat as,

Np = Nicotia na plubaginif olia, N t = Nicotiana tobacum, Mi = Mangoif era, Or = Oryza sativa, P a = Panicum miliaceum,

P s = P isum sativum, Sg = Sauromatum guttatum, St = Solanum tuberosum, T t = T riticum turgidum, V u = V igna unguiculata ,

Zm = Zea mays. Proteins - AA = Asparate aminotransferase, AC = Aconitase, AD = Aldehyde dehydrogenase, ANT = Adenine nucleotidetranslocator, AOX = Alternative oxidase, CcR = Ubiquinol cytochrome C oxidoreductase complex (bc1), CS = Citrate synthase, COX =

cytochrome oxidase, Cyt c1 = cytochrome c1, F1 α , β, δ, and γ = the alpha, beta, delta, epilson and gamma subunits of the ATP synthase, FAd

= d subunit of the FA portion of the ATP synthase, FC = Ferrochelatase, FH = Fumarate Hydratase, FPS = Farnesyl-diphosphate synthase, GD

= Glycine decarboxylase subunits, Gr = Gluathione reductase, HSP = Heat shock protein, HPPK DS = 6-hydroxymethyl 7, 8-dihydropterin

pyrophosphokinase 7, 8 dihydropteroate synthase, ICDH = Isocitrate dehydrogenase, MD = Malate dehydrogenase, ME = Malic enzyme, Mt

= Malate translocator, CI NADH = NADH binding subunit of complex I, PD = Pyruvate dehydrogenase, PPXI = Protoporphyrinagen, PSST

= PSST protein of complex I, RFeS = Rieske FeS protein, RSP = Ribosomal Protein, SHMT = Serine Hydroxymethyltransferase, SOD =

Superoxide dismutase, TOM = Translocase outer membrane, TufM = Translation elongation factor, UCP = Uncoupler protein.



314

Figure 1. Continued.



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for processing [178]. Both domains may overlap. Be-

low, structural properties of the presequences with

relevance for their functions will be discussed.

Targeting properties of presequences

Despite the fact that over 100 plant sequences areavailable, the mitochondrial targeting ability of only

about 10 of these sequences has been demonstrated

experimentally, and detailed analysis has only been

carried out on a handful of sequences. The mitochon-

drial targeting ability of a presequence can be deter-

mined by (1) the use of chimeric constructs containing

the presequence and a marker gene and targeting ex-

amined in vitro and in vivo, (2) the use of synthetic

peptides to inhibit targeting of authentic or chimeric

constructs in vitro and (3) the use of site-directed mu-

tagenesis to test the role of specific residues with in

vitro studies. All approaches have advantages and dis-

advantages. The use of marker constructs is useful

to detect import but in vivo they suffer from the fact

that the nature of the passenger protein may greatly

effect targeting ability. Chloramphenicol acetyl trans-

ferase (CAT) as a passenger protein is targeted more

efficiently than β-glucuronidase (GUS) to plant mito-

chondria [174]. Lack of targeting observed in some

cases may be explained by structural features of dif-

ferent constructs ([207]). Also the proteolytic accessi-

bility in the cytosol of a chimeric construct may vary

greatly with the addition of ‘linker sequences’ [230].

The presequence of the Nicotiana plumbaginifolia

F1β subunit can target passenger proteins to mitochon-dria [30, 32], and although deletions in the C-terminal

region don’t abolish targeting they reduced the ef-

ficiency quite dramatically [32]. This implies that

although the N-terminal portion is sufficient for tar-

geting, the C-terminal region plays a significant role.

Deletion of the N-terminus abolish targeting, and this

is supported with in vitro studies with synthetic pep-

tides derived from the presequences [91]. A similar

functional role for the maize superoxide dismutase

presequence comes from deletion studies [219].

A number of other studies with the adenine nu-

cleotide translocator (ANT) and F1δ presequence re-port that additional residues from the mature region

are required for mitochondrial targeting [100, 136,

223]. Although Mozo et al (1995 [136]) who used

the GUS reporter gene showed that the presequence

of ANT is not sufficient for targeting in vivo, Winning

et al. (1992 [223]) showed that an additional 20 amino

acids from the mature protein did support mitochondr-

ial targeting with dihydrofolate reductase (DHFR) as

a marker. It cannot be concluded that the N-terminal

region of the adenine nucleotide translocator does not

play a role in mitochondrial targeting. Even though

mature ANT can be imported to the mitochondrion

[223], a study with chimeric precursors containing

both mitochondrial and chloroplast targeting peptidesshowed that the first sequence (most N-terminal) ex-

erted the targeting information [174]. Therefore the

N-terminal presequence of the ANT may play a role

in targeting in vivo.

Site directed mutagenesis studies of the prese-

quence of the soybean alternative oxidase have been

carried out to elucidate the targeting requirement of

a plant mitochondrial precursor protein (Tanudji et

al., unpublished). Approximately 30 different mu-

tants were constructed to elucidate the role of indi-

vidual residues. It was found that arginine residues

throughout the presequence were important for target-

ing ability. However, not just arginine (or positive)

residues in the region of the predicted amphiphilic α-

helix were important, but arginine residues near the

processing site also played an important role in tar-

geting. Changing two arginine residues at the −2 and

−10 region of the presequence (outside the predicted

helical region) inhibited import as much (80%) as

changing two arginines (−20, −30) predicted to be in

the helix forming region. Additionally a region of the

presequence that contained the putative amphiphilic

α-helix was not sufficient to support efficient import

into isolated soybean mitochondria (Tanudji et al., un-

published). This study would indicate that the entirepresequence is important for targeting, which is in

general agreement with the deletion studies for the

F1β if the amount of import is examined. It does also

indicate that an amphiphilic α-helix alone is not the

only requirement for an efficient targeting to plant mi-

tochondria. It will be of interest to investigate if the

in vitro results are consistent with the in vivo studies

using chimeric constructs.

Sorting of precursors between mitochondria and

chloroplasts

Mitochondrial and chloroplastic precursor proteins

were shown to interact in the cytosol with molecular

chaperones, heat-shock proteins of 70 kDa (HSP70s)

(for review see Hartl, 1996), mitochondrial import

stimulation factor (MSF) [72] and also a presequence

binding factor (PBF) [137]. These proteins seem to

co-operate in binding to newly synthesised precursor



319

proteins, assist their folding, prevent aggregation [67],

keep them in a transport-competent form (unfolded

or loosely folded) and convey them to import recep-

tor complexes on the surface of the organelle [190].

Despite the fact that there exist distinct structural dif-

ferences in mitochondrial and chloroplastic targeting

peptides [201] it is possible that cytosolic factors thatinteract with the targeting peptides contribute to the

specificity of protein targeting and/or to prevention of

miss-sorting [121].

A number of studies demonstrated that mitochon-

drial protein import in plants was highly specific, i.

e. mitochondria appear to import precursors of mi-

tochondrial proteins only, while chloroplasts appear

to import precursors of chloroplast proteins only [18,

214]. Using a homologous in vitro organelle import

system, i.e., isolated spinach mitochondria and chloro-

plasts, we have shown that import was specific for both

organelles [214]. Also, recently, specific targeting into

isolated mitochondria and chloroplasts has been re-

ported for proteins from C. reinhardtii [144]. The

C. reinhardtii precursors of the F1α subunit and the

Rieske FeS protein were imported into C. reinhardtii

mitochondria with high efficiency in a membrane po-

tential dependent manner. The C. reinhardtii F1β sub-

unit, containing a C-terminal extension in addition to

the N-terminal presequence, was imported with much

lower efficiency. Also, the import of mitochondrial

heterologous precursor proteins from higher plants,

soybean alternative oxidase and the N. plumbaginifo-

lia F1β subunit, was much less efficient. A number of

studies using transgenic approaches also showed highspecificity of targeting into plant mitochondria. The

chimeric constructs consisting of the presequence of

N. plumbaginifolia F1β coupled to CAT or glutamine

synthase were specifically targeted into mitochondria

[18, 83].

Mis-sorting of proteins has also been reported in

a few cases. Hurt et al. (1986 [194]) reported that

the RuBisCO transit peptide from C. reinhardtii could

direct proteins, mouse DHFR and yeast cytochrome

oxidase subunit IV, into yeast mitochondria. However,

the system was non-homologous and the chloroplast

transit peptides from C. reinhardtii were later reported

to contain an amphiphilic α-helix, characteristic of mi-

tochondrial targeting peptides [63]. Huang et al. (1990

[89]) showed that the mitochondrial presequence of

the yeast cytochrome oxidase subunit Va can function

both as a mitochondrial and chloroplastic targeting

peptide in transgenic tobacco. However, it was con-

cluded that this ‘mis-targeting’ did not represent a

physiological pathway, as in vitro studies showed that

the import of this chimeric construct occurred at zero

degrees and was not receptor mediated. Both these

cases of mis-sorting are examples of mis-sorting in

heterologous systems. However, Criessen et al. (1995

[37]) have reported that the pea glutathione reduc-

tase precursor protein is directed both to chloroplastsand to mitochondria in transgenic tobacco. The PsaF

protein from C. reinhardtii but not the PsaK protein

has been shown to be imported in vitro into spinach

mitochondria and also recently into C. reinhardtii mi-

tochondria, in vitro [92, 144]. However, characteristics

of the import process were peculiar. This process was

shown to be independent of the presequence, as a

mutant protein devoid of presequence was protease-

protected upon incubation with spinach mitochondria.

Furthermore, the protease-protection also appeared

to be independent of mitochondrial import receptors,

as it was not inhibited by a synthetic peptide corre-

sponding to a mitochondrial presequence [92]. The

transit peptides of two chloroplast envelope proteins,

the triose-3-phosphoglycerate phosphate translocator

(TPT) and a 37 kDa protein of unknown function, have

the ability to form an amphiphilic α-helix, a feature

considered to be essential for mitochondrial but not

for chloroplastic targeting sequences [28, 201]. Import

studies of these proteins showed that they interacted

with yeast mitochondria in a receptor dependent man-

ner [28]. Chimeric constructs containing TPT transit

peptide and 5 or 23 amino-terminal residues of the

mature TPT coupled to CAT were also imported into

plant mitochondria in vitro [175]. However, in vivo intransgenic tobacco, the construct containing the transit

peptide and 5 residues of the mature TPT was found

in the cytosol, whereas, the construct containing 23

residues was specifically imported into chloroplasts.

These studies clearly show that the import process has

more stringent specificity in vivo [175].

In summary, the above described results indi-

cate that some chloroplast transit peptides contain

sufficient information for specific interaction with

mitochondrial import receptors, however, they also

show that import specificity between mitochondria

and chloroplasts is maintained in vivo. Interestingly, it

has been reported that transit sequences of chloroplast

precursor proteins but not mitochondrial or peroxi-

somal precursors are phosphorylated by a plant spe-

cific cytosolic protein kinase and that phosphorylated

precursors bind to chloroplasts [205]. Dephosphory-

lation seems to be required to complete the precur-

sor translocation process across the membranes. This



320

phosphorylation-dephosphorylation cycle of chloro-

plast destined precursor proteins might represent one

of the events involved in a specific sorting process

between mitochondria and chloroplasts [205]. If an

amphiphilic α-helix is not the only requirement for

plant mitochondrial import, the few chloroplast pre-

cursor proteins that do contain such a motif in thetransit peptide may only be targeted to plant mitochon-

dria quite inefficiently. Along with other factors that

may contribute to targeting specificity such as chaper-

ones [121] it seems that targeting specificity is quite

high. What the studies on specificity do indicate is

the possible artefacts of using heterologous systems

with chimeric constructs as the nature of the passen-

ger protein and it’s susceptibility to various proteases

may differ between the authentic construct and the

chimeric construct used in import studies ([174, 207,

230]) Tanudji et al., unpublished).

Protein import machinery

Components of the outer membrane translocase

Several components of the mitochondrial import ma-

chinery (translocase) of the outer membrane (Tom)

have been identified in plants, potato (p) Tom 20

[81], Vicia faba (Vf ) Tom 40 [152] and outer mem-

brane HSP 70 [133]. Additionally, mtHSP 70 [209]

has been cloned and expressed sequence tags exist for

Tim 17 from Arabidopsis and rice, and Tim 23 from

Arabidopsis.Tom 20 from potato has been cloned, it has a pre-

dicted molecular mass of 23 kDa. It displays only

20% identity with other Tom 20 but similarity values

rise to 50% and it is also proposed to contain a tetra-

tricopeptide repeat (TPR), a characteristic of many

components of the mitochondrial import machinery

[12, 13, 81]. Preincubation of mitochondria with an-

tibodies to pTom 20 inhibited the import of a variety

of precursors by 30% to 40%. The lack of complete

inhibition is not unexpected as similar studies in yeast

suggest several receptors with overlapping specifici-

ties [161]. pTom 20 is imported into mitochondriawithout the need for a protease (trypsin) sensitive

receptor on the outer membrane [81]. The character-

isation of Tom 20 from plants represents an important

step in understanding the import process as further

studies with this components may help to understand

how specificity of targeting is maintained. For in-

stance when h Tom 20 was cloned and overexpressed

in yeast (y) it was shown that it could complement

a y Tom 20 mutant. However, in contrast to the y

Tom 20 it did not support import of artificial precur-

sors under the influence of a ‘cryptic’ mitochondrial

targeting sequence [128]. This indicates that similar

components within different phylogenetic groups may

display substantially different properties.A Tom 40 component has been identified from Vi-

cia faba, with an apparent molecular mass of 42 kDa.

This protein was identified with antibodies raised

against Neurospora crassa ( Nc) Tom 38 and y Tom

40 (formally ISP 42) [152]. These antibodies inhib-

ited import of several precursor proteins into isolated

mitochondria. This component is proposed to be one

of the central proteins of the general insertion pore in

fungi. However, no further information of its identity

or function is currently available in plants. The other

component of the outer membrane that has been listed

is the outer membrane (OM) HSP 70 [133]. No role

in the import process has been shown for this com-

ponent. It is not present in N. crassa or yeast but a

similar component has been reported from mammals

[122]. As different HSP 70 isoforms play several roles

in the import process it is interesting to speculate that

the OM HSP 70 may also be involved in the import

process in higher organisms. It is possible that pre-

cursors proteins are passed from cytosolic HSP 70 to

the OM HSP 70 before binding to the outer membrane

receptors.

Import pathways

Three import pathway that have been elucidated for

plant mitochondria are outlined in Figure 2. The sim-

plest pathway exists for p Tom 20 (pathway 1) which

does not require a trypsin sensitive components on the

outer membrane [81]. The import conditions used in

this study employed both ATP and substrates to gener-

ate a membrane potential so the involvement of these

components cannot be ruled out. However, in yeast, a

membrane potential is not required for the import of

outer membrane proteins [118].

The third import pathway outlined in Figure 2 may

be described as the general import pathway [141].The import of proteins in this pathway requires both

extra- and intramitochondrial ATP, cytosolic factors,

outer membrane proteinaceous receptor(s); precursor

proteins that use this pathway are usually processed

by MPP (non-cleaved precursors may also use this

pathway). It is likely that this pathway uses other com-

ponents such as Vf Tom 42, Arabidopsis thaliana ( At )



321

Figure 2. Diagrammatic representation of three pathways of protein import into plant mitochondria. Pathway 1 shows the import of an outer

membrane protein (Tom 20) which inserts spontaneously into the outer membrane [81]. Pathway 2 shows the import of the ATPsynthase

FAd subunit precursor protein from soybean. This precursor protein is synthesised with an N-terminal presequence which interacts with the

outer and inner membrane translocation machinery components. This precursor protein does not use ATP-dependent cytosolic factors for

targeting through the cytosol [44]. Pathway 3 shows the import of the soybean alternative oxidase precursor protein. This precursor protein is

also synthesised with an N-terminal presequence and requires ATP-dependent cytosolic factors. Import via pathways 2 and 3 converges with

the requirement for a membrane potential across the inner membrane, and ATP in the matrix for driving the inner membrane translocation

machinery [44]. Components of the inner and outer translocation machinery which have been identified in plants are labelled in larger lettering.

HSP70, Tom 20 and Tom 40 [81, 133, 152] on the outer membrane, Tim 23, Tim 17 ( Arabidopsis thalianaEST database), and mtHSP 70 [202],

in the inner membrane and matrix. Other components labelled in smaller lettering which have been identified in fungi are shown. Components

involved in processing and assembly have not been shown.

Tim 17 and Tim 23 but since the specific involve-

ment of these components has not been experimentally

demonstrated in plants they will not be further dis-

cussed. The requirement for external ATP in this

pathway may come from two sources, cytosolic HSP

70 and/or an additional factor. Although it was previ-

ously thought that cytosolic HSP 70 had a requirement

for ATP, characterisation of the mitochondrial import

stimulating factor (MSF) from rat using yeast mito-

chondria indicates that it is MSF that has the obligate

ATP requirement and that precursors bound to cytoso-

lic HSP 70 may be imported without ATP [73, 74,104]. However, this has only been shown with the

adrenodoxin precursor and it has not be applicable

to all precursors using the general import pathway.

Additionally, it should be remembered that rat mito-

chondria contain an outer membrane HSP 70 that is

absent in yeast and the use of a heterologous system

may not give a clear picture. In vivo, assuming that

cytosolic HSP 70 would bind to precursor proteins and

that ATP would be present, it is likely that ATP is used

by cytosolic HSP 70 in the import process.

There is evidence that another cytosolic factor in

addition to cytosolic HSP 70 is required in the plant

mitochondrial import pathway. This comes from stud-

ies using wheat germ as a translation lysate rather

than rabbit reticulocyte lysate, the assay used to iden-

tify and purify MSF [131]. Mitochondrial precursors

translated in the wheat germ translation system were

imported into mitochondria at a very low efficiency.Addition of rabbit reticulocyte lysate stimulated im-

port. This factor was shown to be N-ethylmaleimide

(NEM) sensitive and required ATP, similar to purified

MSF from rat liver. It has been shown that import

of the soybean FAd precursor into soybean mitochon-

dria could be supported by the wheat germ alone [44].



322

Significantly this import did not require external ATP

and was not sensitive to NEM treatment of the precur-

sor. This implies that it did not require this additional

factor, that is either inactivated or absent in wheat

germ. Additionally, protease treatment of mitochon-

dria showed that import of the FAd precursor used a

less protease sensitive protein component on the outermembrane, indicating that a different receptor may be

involved [44]. Therefore, an additional import path-

way (pathway 2, Figure 2) has been proposed to exist

in plant mitochondria (Figure 2).

Evidence suggests that import pathways 2 and 3

converge at a common point for translocation into mi-

tochondria. This is supported by the fact that both

pathways require a membrane potential and that pre-

cursors are processed by MPP ([44], unpublished

data). The translocation force for import is postulated

to be derived from Tim 44 in combination with mtHSP

70, although it is still unclear whether a Brownian

ratchet or translocation motor model is the mecha-

nism of import [72, 86]. In plants mtHSP 70 has been

cloned from a number of sources (Figure 1) but no

direct experimental evidence exists for its role in im-

port. In spinach, we have shown that mtHSP 70 is

partially associated with the mitochondrial inner mem-

brane [202]. It is likely that it fulfills a similar function

as in yeast and the proposal that mtHSP 70 may regu-

late import in plants is discussed in section on control

of protein import.

Mechanism of translocation

Import (translocation) of proteins into mitochondria is

inhibited by components that abolish the membrane

potential such as the ionophore valinomycin, and un-

couplers CCCP and FCCP [30, 77]. Using a potassium

driven membrane potential it has been concluded from

studies in fungal systems that it is the ψ compo-

nent of the membrane potential that is required [125,

153]. No specific studies in plants have addressed this

question.

A variety of respiratory substrates as malate, succi-

nate, glycine and NADH have been shown to support

precursor import into plant mitochondria in varioussystems [31, 49, 102, 203, 209, 212]. We have com-

pared the efficiency of each of these substrates to

support import with soybean mitochondria. We have

also investigated if respiration via the alternative path-

way alone can support import. We have found that

succinate and then NADH support import best of two

different precursors. Added NADH is only oxidised

via the external NAD(P)H dehydrogenase [48, 132,

134, 198], and can support import via the alternative

pathway alone. It indicates that a sufficient membrane

potential can be generated in the absence of any proton

translocation via the well characterised complexes of

the cytochrome chain [43, 206].

It is puzzling why import should differ with dif-ferent substrates if only a low membrane potential (40

to 60 mV) is required as suggested from the studies

in fungal systems [125, 153]. Using succinate as a

substrate to support import, a competitive inhibitor

to complex II, malonate, we could clearly demon-

strate that the import of the FAd was inhibited with

10 mM malonate, whereas the alternative oxidase was

not fully inhibited with 50 mM inhibitor. This strongly

suggests a difference in the magnitude of the mem-

brane potential required for the import of these two

precursor proteins [43]. A different threshold of mem-

brane potential has been reported to be required for

different precursors in fungi [125]. This study showed

that the requirement for different magnitude of the

membrane potential corresponded to a property of the

presequence, and was not a consequence of the length

of the mature protein. It was proposed that the greater

number of positive residues in the presequence the

lower the requirement for a membrane potential. As

both the alternative oxidase and FAd precursor have

five positive residues this cannot be the only factor

that causes the difference seen with these two pre-

cursors. An explanation for the difference may be the

nature of the mature protein. The insertion of the pre-

sequence into and across the inner membrane seemsto be a reversible event [141]. However, the presence

of a hydrophobic segment may stabilise the translo-

cation intermediate [71]. As the FAd protein is very

hydrophilic [181], the difference in the requirement

for a membrane potential may lie in the fact that a

higher threshold is required to maintain the prese-

quence of the FAd precursor on the inside of the inner

membrane compared to the alternative oxidase. The

latter is a transmembrane protein and once its pre-

sequence has inserted across the innermembrane the

hydrophobic segments may directly insert into the in-

ner membrane or stabilise this precursor. Considering

the branched nature of the respiratory chain in plant

mitochondria, close examination of the membrane po-

tential requirement with a variety of precursors and

chimeric constructs is warranted, measuring both im-

port and membrane potential under similar conditions,

to understand the translocation of proteins to various

locations within the mitochondrion.



323

The ψ component of the membrane potential is

proposed to exert its effect via an electrophoretic ef-

fect on the positively charged presequence [77]. This

is supported by a study which elegantly showed that

import of a fusion protein containing the targeting do-

main of Tom 70 (and DHFR) can be inserted into the

inner membrane of ruptured rat liver mitochondria in aψ dependant manner, but removal of the three posi-

tive signals in the targeting domain, results in insertion

into the inner membrane that becomes independent of

ψ [129].

A second role for the membrane potential has

emerged with the characterization of the import ma-

chinery. It has been demonstrated that Tim 23 acts

as a voltage sensor, undergoing dimerization in the

presence of a membrane potential, the extent of dimer-

ization being dependant of the extent of the membrane

potential. Binding of the matrix targeting domain of a

presequence dissociates the dimer opening the import

channel [6]. The two different roles for the membrane

potential are not contradictory as it would appear

from the studies showing that the threshold of mem-

brane potential for dimerization [6] is lower than that

required for the electrophoretic effect on precursors

[125]. This issue, however, has not been addressed in

the same study.

Another factor that may affect protein import

is redox status of SH-groups of the inner mem-

brane translocase. We have investigated the effect of

sulfhydryl group reagents on import of the in vitro

transcribed/translated precursor of the F1β subunit

of the ATP synthase (pF1β) into Solanum tubero-sum mitochondria [203]. We have used a reducing

agent, dithiothreitol (DTT), an alkylating membrane-

permeant, N-ethylmaleimide (NEM), a non-permeant,

3-(N-maleimidopropionyl)-biocytin (MPB), an SH-

group specific agent 5,5-dithiobis-(2-nitro-benzoic

acid) (DTNB) as well as an oxidising cross-linker,

copper. DTT slightly stimulated the mitochondrial

protein import, whereas NEM and Cu2+ were in-

hibitory. Inhibition by Cu2+ could be reversed by

addition of DTT. We have dissected the effect of the

SH-group reagents on binding, unfolding and trans-

port of the precursor into mitochondria. The efficiency

of receptor-mediated binding of pF1β to mitochon-

dria increased in the presence of DTT, NEM or Cu2+.

This demonstrated that the inhibitory effect of NEM

and Cu2+ on the efficiency of import was not due to

the interaction of the SH-group reagents with import

receptors. Modification of pF1β with NEM prior to

the import resulted in stimulation of import, whereas

no effect was seen with DTT and Cu2+. It h as

been reported that treatment of the alternative oxi-

dase precursor with 5 mM NEM inhibits its import

into mitochondria. This was proposed to be due to

inactivation of an NEM sensitive factor [44]. Import

of pF1β through a receptor-independent bypass-route

and into mitoplasts was sensitive to DTT, NEM andCu2+ in a similar manner as import into mitochondria.

Both DTNB and a membrane-impermeant, MPB were

shown to inhibit protein import into mitoplasts. These

results taken together indicate that protein import into

plant mitochondria is inhibited by modification of the

SH-groups of the protein import machinery located on

the outer surface of the inner mitochondrial membrane

[203].

A question that arises with regard to the translo-

cation process in plant mitochondria is the timing of

the processing event with respect to the location of

the precursor. Considering the membrane location of

MPP (next section), the question arises if precursors

are processed during or after translocation. The latter

would require that matrix located proteins would have

to come into close proximity with the inner membrane

after import whereas the former would require that the

cytochrome bc1 complex would be physically close to

the import site. It has been reported that characteris-

tic inhibitors of MPP, EDTA and orthophanenthroline,

inhibit import into plant mitochondria, in contrast to

the situation in fungal systems [216]. Metal chelators

which could not pass the inner membrane did not in-

hibit import indicating existence of a metal dependant

translocation step on the inside of the inner membrane.Further characterisation of the Tim components of the

import machinery may help to answer this question

by using immunolocalisation or immunoprecipitation

techniques.

It can be concluded that the current understanding

of the translocation process in plants is at an early

stage and heavily relies on studies from yeast sys-

tem to interpret the available experimental data from

plant systems. Although some of the components have

been identified, functional characterisation still needs

to be carried out with a variety of precursor proteins

as it cannot be presumed that these components have

the same properties in plants as they have in simpler

fungi. The case of h Tom 20 compared to y Tom 20

exemplifies the need for caution in trying to fit exper-

imental data from one system with the characterised

components of another system [128]. The other area

that needs to be further investigated with reference to

the translocation mechanism is the involvement and



324

consequences, if any, of the integrated MPP into the

cytochrome bc1 complex. Although studies with puri-

fied MPP (see below) indicate no link between elec-

tron transport and processing it would be of interest to

test this out with intact mitochondria.

Mitochondrial protein processing

The mitochondrial processing peptidase (MPP) re-

moves the amino-terminal targeting signal, the prese-

quence, from nuclear-encoded mitochondrial precur-

sor proteins, during or shortly after protein import into

mitochondria [77, 166]. A striking feature of MPP

is that it is a general peptidase, as it acts on several

hundred mitochondrial precursor proteins, yet MPP is

specific as it recognises a distinct cleavage site and

specifically cleaves off the presequences, that show

low sequence similarity, in one single cut. Proteolytic

removal of the targeting peptide is not essential for im-

port [94, 228, 232], Sjöling, unpublished), but might

be necessary for proper folding of the protein [162].

The subunits of the MPP were the first geneti-

cally identified components of the import machinery

[228]. It consists of two structurally related subunits,

α-MPP and β-MPP, which co-operate in processing.

Both subunits are essential for processing. In plants,

the MPP has been purified from potato tubers [21]

spinach leaves [56, 60] spinach roots (Sjöling, unpub-

lished) and wheat [26]. The plant MPP was shown to

reside in the inner mitochondrial [59] integrated into

the cytochrome bc1 complex of the respiratory chain[21, 54, 60]). Thegenes encoding the potato MPP have

been cloned [21, 53, 55]) and shown to be homologous

to each other.

In mammals and yeast both subunits are soluble

in the matrix as a heterodimer, whereas in N. crassa,

the matrix subunits are isolated as monomers [79, 148,

229] and 70% of β-MPP can be found associated to

the mitochondrial inner membrane and it is identical to

the core 1 protein of the bc1 complex of the respiratory

chain [171].

Early studies of plant mitochondrial protein

processing showed that detergent extracts of lysedcauliflower and Vicia faba mitochondria could cleave

N. crassa precursors to mature proteins [214] and that

mitochondrial extracts were unable to cleave chloro-

plast precursors [213]. The processing activity of plant

mitochondria was found in the membrane fraction [59]

and could not be dissociated from the membrane by

high pH, high ionic strength or chaotropic reagents.

Addition of the soluble fraction, the matrix, did not

affect the activity of the membrane fraction.

The plant cytochrome bc1 complex has a simi-

lar subunit composition as the corresponding enzymes

of mammals and fungi. It comprises 10 polypeptides

ranging in molecular mass from about 60 to 8 kDa [21,

56], including the Rieske-FeS protein, cytochromesc1 and b. The exception is the apparent existence, on

SDS-PAGE, of three to four core proteins, instead of

two, as found in the bc1 complex of mammals and

N. crassa [165, 210]. Three core proteins have been

observed in the bc1 complex of spinach leaves, beet

[11, 21] and yeast [123,191]. There is even the appear-

ance of four core proteins in the bc1 complex of potato

wheat [26] and spinach roots (Sjöling, unpublished).

It was suggested that these extra polypeptides, which

could be seen on SDS-PAGE, were due to the exis-

tence of incompletely processed core precursors [123,

191]. The polypeptides, however, were always present

in equimolar ratio. Therefore, the existence of more

than two core proteins is rather the result of occurrence

of isoenzymes. Blue native gels show a composi-

tion of potato bc1 complex with the same molecular

mass as bovine bc1 complex, indicating that the potato

bc1 complex consists of only two core proteins per

monomer [96]. Immunoprecipitation using antibody

against one of the isoforms in potato reveal an enzyme

complex containing only two core proteins and gene

specific oligonucleotides reveal that the genes encod-

ing α-MPP of potato are differently expressed in all

tissues analysed but transcript levels do not vary be-

tween tissues [96]. The core proteins of the spinachbc1 complex were immunological [56, 60] and by se-

quence analysis [21] identified as MPP subunits [21].

The genes for the isoproteins have probably arisen

by gene duplication followed by sequence divergence

[47]. The biological significance of these isoforms re-

mains to be determined. Possibly they act as a buffer

against detrimental mutations effecting the highly ex-

pressed genes or it is the result of the polyploidy of

cultured plants [55, 96].

Characteristics of the MPP/bc1 complex

The molecular mass of the MPP/bc1 complex cor-

responds to a dimer of the bc1 complex [57]. The

dimeric form of the bc1 complex has been observed in

other species by chromatography, electron microscopy

and X-ray crystallography [150, 168, 210, 227]. It is

not known if the monomeric forms in the dimer co-

operate in the respiratory electron transfer neither if



325

the dimeric form is essential for peptidase activity in

plants.

The plant MPP is active over a broad pH range,

pH 6–11 (with optimum at pH 8) and over a broad

temperature range, 10–50 ◦C (with optimum at 35 ◦C).

The plant MPP is a metallopeptidase. This is in ac-

cordance with MPP from other organisms [14, 127].There is no effect on the processing activity by in-

hibitors of the serine-, cystein-, amino- aspartic- or

thiol-type protease. On the other hand, the metal

chelators, ortho-phenantroline and EDTA, totally in-

hibit the processing activity. The processing activity is

not dependent on exogenous metal ions [56, 57] but it

is slightly stimulated by the addition of Ca2+, Mn2+,

or Mg2+. Co2+, Fe2+, Zn2+, Cu2+ or Ni2+ inhibit

the peptidase activity [58, 66, 177]. It has been shown

that inhibition by heavy metals, or transition metals,

is a common feature amongst many zinc containing

metallo-peptidases [35, 115]. In reconstitution exper-

iments with spinach apo-MPP, prepared by dialysis

against ortho-phenantroline and then buffer to remove

the chelator, the activity could be restored by the addi-

tion of Mn2+, Zn2+ and partially by Mg2+, Co2+ and

Cu2+ [58]. Mn2+, Mg2+, Co2+, and Cu2+ are divalent

cations frequently found to substitute for zinc in met-

allopeptidases [4]. Particle Induced X-ray Emission

(PIXE), Inductively Coupled Plasma-Atomic Emis-

sion Spectroscopy (ICP-AES) and Total Reflexion

X-ray Fluorescence (TRXE) show that the spinach

MPP/bc1 complex contains iron, copper, calcium and

zinc [58]. High ionic strength inhibits the spinach

leaf MPP activity [56] at higher concentrations (>0.1 m), however, it does not dissociate the spinach core

proteins from the bc1 complex. Surprisingly, KCl con-

centrations above 1M stimulate the processing activity

of potato MPP [53].

As the plant cytochrome bc1 complex is involved

in both protein processing and respiration, one may

wonder whether protein processing is dependent on,

or affected by, the redox state of the bc 1 complex or

on respiration. Complete oxidation or reduction of the

redox centra of the bc1 complex in sub-mitochondrial

particles (SMP) in the presence of respiratory sub-

strates and respiratory chain inhibitors has only slight

or non effect on the processing activity. Reduction of

the bc1 complex with DTT or dithionite diminishes

the processing activity to about 50%, probably by

effecting metal/ligand interaction. In summary, even

though there is a bifunctionality of the oligomeric

MPP/bc1 complex, there is no correlation between

electron transfer and protein processing in vitro [56].

Soluble plant MPP

Preliminary characterisation of the processing prop-

erties of plant mitochondria has shown that plants

contain a processing peptidase with similar proper-

ties to fungal MPP [213, 214]. Despite the fact that a

processing activity can be clearly located and isolatedfrom the mitochondrial inner membrane, a process-

ing activity located in the matrix fraction was also

reported from Vicia and spinach as earlyas 1992 and in

subsequent studies, although it was not characterised

in detail [59, 80, 103]. We showed that the matrix

located activity was not due to an ATP dependent

protease as it was not dependent on ATP. Likewise,

there was no evidence for non-specific breakdown

such as smearing or appearance of other breakdown

products on SDS-PAGE [103]. Additional evidence

for the location of a processing peptidase in the matrix

of plant mitochondria came from the studies in which

the purified N. crassa β-MPP subunit restored the

processing activity in solubilized plant mitochondria

which were immuno-depleted with antibodies against

N. crassa β-MPP and unable to catalyse the process-

ing activity [214]. This is in contrast to studies of the

now extensively characterised membrane located MPP

from plants which have shown that the processing ac-

tivity cannot be separated from the cytochrome bc1

complex.

In a combined effort from four laboratories, we re-

investigated the possible presence of a matrix-located

processing activity in plant mitochondria, in terms

of its ability to generate mature size products fromprecursor proteins, its specificity, and its inhibitor

sensitivity [186]. We have used three mitochondrial

precursor proteins and two plant species. We inves-

tigated occurrence of an additional, matrix located

processing activity, by incubation of the precursors

of the soybean mitochondrial proteins, alternative ox-

idase, the FAd subunit of the ATP synthase and the

tobacco F1β subunit of the ATP synthase, with the

membrane and soluble components of mitochondria

isolated from soybean cotyledons and spinach leaves.

A matrix-located peptidase specifically processed the

precursors to the predicted mature form in a reactionwhich was sensitive to o-phenanthroline, a characteris-

tic inhibitor of MPP. The activity was also inhibited by

NEM, an inhibitor of fungi MPP. The specificity of the

matrix peptidase was illustrated by the inhibition of

processing of the alternative oxidase precursor in both

soybean and spinach matrix extracts upon altering a

single amino acid residue in the targeting presequence



326

(−2Arg to Gly). Additionally, there was no evidence

for general proteolysis of precursor proteins incubated

with the matrix. The purity of the matrix fractions was

ascertained by spectrophotometric and immunologi-

cal analyses. The results demonstrate that there is a

specific processing activity in the matrix of soybean

and spinach corresponding to 50% and 20%, respec-tively, of the total mitochondrial processing activity.

The identity of the matrix peptidase is not known.

In a lower eukaryotic photosynthetic organism C.

reinhardtii, in vitro processing studies revealed that

in contrast to the situation in higher plants, and in

accordance with studies in yeast and mammals, the

processing of the precursors was catalysed by a matrix

located peptidase and not by a peptidase integrated

into the cytochrome bc1 complex of the respiratory

chain [144]. Interestingly, maturation of the homolo-

gous precursors inside mitochondria required addition

of ATP during import. In the absence of ATP, pre-

cursors were protease-protected inside mitochondria,

but most of the imported proteins existed in a non-

processed form, indicating requirement for ATP for

import of the precursor into the matrix.

The different location of MPP in various organisms

is proposed to represent divergence from a single orig-

inal evolutionary event [27]. This is in agreement with

a monophyletic origin for mitochondria [68].

A new family of metallo-endopeptidases

Sequence alignment shows that the MPP subunits of potato, rat, yeast, N. crassa and the core proteins

of the bc1 complexes, share sequence similarity with

the pitrilysin family [156–158]. The pitrilysin family

includes the pitrilysins from E. coli, the insulin de-

grading enzymes from mammals and Drosophila, the

N-arginine dibasic convertase from rat and a couple

of additional enzymes. Pitrilysins are highly specific

metallo-endopeptidases, and like the MPP, recognise

their substrates without defined amino acid residues

around the scissile bond, showing that recognition is

on the basis of higher order structure rather than of

the amino acid sequence [1, 8]. By site directed muta-genesis, an inverted zinc-binding site was discovered

that was used to define a new family of metallo-

endopeptidases, the pitrilysin family [5, 7, 9, 151].

A glutamate in this zinc binding motif possibly has

the function as electron donor for a hydrogen bond

to a water molecule which attacks the carbonyl at the

peptide bond to be cleaved [29]. The HXXEH74−76E

signature is conserved in all β-MPPs but degenerate in

α-MPPs and core proteins.

Substrate specificity of processing

How does the MPP recognise such a diversity of mito-

chondrial precursor proteins? What features determinethe MPP cleavage? What are the structural features

of the precursor protein that are recognised by the

MPP? Is the sequence of a few amino acids around

the scissile bond sufficient or do other structural el-

ements contribute to the recognition of the cleavage

site? These questions have been addressed using sev-

eral approaches, including statistical analysis of plant

presequences for common features, in vitro studies of

affinity of chemically synthesised targeting peptides

for MPP, structural analysis of presequence peptides

and site directed mutagenesis of precursor proteins.

Except for the common cleavage motifs R-3 and

R-2, [64, 169, 178, 201] no consensus sequence has

been identified within the presequences. Since there is

a significant amount of variability in the presequence

among different precursors, we and others have pro-

posed that MPP recognises higher order structural

features [93, 109, 176, 178, 179, 208], possibly in

conjunction with basic residues [84, 88] rather than a

specific amino acid sequence. Although the local argi-

nine motifs, R-3, R-2, represent parts of the features

enhancing precursor processing, the motifs are highly

degenerate and can even be found elsewhere in the

precursor protein since arginine residues are present

at multiple positions in all proteins. MPP, however,does not cleave at the other sites, making it obvious

that the R-3 or R-2 motifs are not sufficient for spe-

cific cleavage, but that additional common features are

required.

Studies have shown that synthetic peptides corre-

sponding the C-terminal portion the presequence of

the F1β ATPase subunit of N. plumbaginifolia inhibit

processing of pF1β [176] to a higher extent com-

pared with an N-terminal peptide. These results show

that the C-terminal peptide has higher affinity for the

spinach MPP and that MPP recognises the C-terminal

domain rather than the N-terminal domain of the pF1βpresequence. This is consistent with results of studies

of alignment of plant presequences. In those studies

several presequences were found to contain conserved

N-terminal and C-terminal domains. This may have

functional importance, the N-terminal domain being

important for targeting and import and the C-terminal

domain for interaction with MPP [178]. Shorter pep-



327

tides corresponding to 4, 6 and 11 amino acid residues

relative to the cleavage site of the pF1β presequence

had no significant inhibitory effect on processing. This

could indicate either that a peptide longer than 11

amino acid residues is required for interaction with

MPP, or that these shorter peptides lack a feature im-

portant for interaction with MPP, which is presentin the longer C-terminal peptide, or both. Interest-

ingly, by secondary structure prediction [176] and

circular dichroism analysis (Sjöling, unpublished) the

longer pF1β C-terminal peptide was shown to contain

a helical element which is not present in the shorter

C-terminal peptides. The helical structure is proposed

to facilitate interaction of the precursor with MPP.

Similar results were shown with the soluble rat liver

MPP [179], indicating that both the membrane bound

plant MPP and the soluble mammalian MPP recognise

similar higher order structure

In addition, a mutant peptide corresponding to the

22 C-terminal residues of the pF1β presequence with

a reduced helix (through the substitution of a serine

for a proline residue which brakes the helix) does

not have the same affinity for spinach MPP as a 22

residues long C-terminal wild-type peptide containing

the intact helix (Sjöling, unpublished). Disrupting the

same C-terminal helix of pF1β by site-directed muta-

genesis of the presequence of the complete precursor

protein inhibits processing (Sjöling, unpublished). It is

possible that a C-terminal helix of the presequence fa-

cilitates the interaction with MPP through helix/helix

packing with the peptidase, or that the helical segment,

followed by a rather loose structural part of the ma-ture chain, constitutes a transition point functioning

as recognition element for MPP as suggested by von

Heijne [201].

The MPP of differentorganismsresides in different

mitochondrial suborganellar compartments. Results

show that despite the fact that the plant MPP is in-

tegrated into a membrane-bound oligomeric complex

in vivo, whereas the mammalian MPP is soluble, the

recognition of the substrate is conserved, and the same

structural features upstream of the cleavage site are

recognised by both the spinach and the rat MPP [179,

208]. In addition, results also show that the presence of

a typical mitochondrial cleavage site is not always suf-

ficient for processing by either soluble or membrane

bound MPP.

The necessity of basic amino acid residues within

the presequence for the targeting of mitochondrial pre-

cursors to the mitochondria has been accepted [2, 76,

77] however, the role of basic residues for MPP cleav-

age is not understood. Mutagenesis studies of both

mammalian [2, 142, 146, 182] and plant precursor

proteins (Tanudji et al., unpublished, Sjöling, unpub-

lished) suggest that basic residues at proximal as well

as distant positions upstream relative to the cleavage

site are important for specifying the cleavage. An argi-

nine residue is suggested to be required for the peptidebond cleavage and possibly for docking the precursor

at the correct site for MPP cleavage [149]. According

to the cleavage site classification by a self organising

network [169] the role of arginine residues seems to be

similar in different organisms, although location and

topology of MPP varies in different organisms.

Mutational analysis of plant presequences

The specificity of protein processing has been

analysed by site directed mutagenesis of prese-

quences. Possibly the recognition site for MPP is

physically separate from the actual cleavage site in

the precursor protein, as has been suggested by Bed-

well and co-workers [10]. The role of both proximal

and distal arginine residues and secondary structure

for processing has been investigated using the soybean

alternative oxidase and the tobacco F1β precursor.

The arginine residue at position −2 from the cleav-

age site of the alternative oxidase presequence, which

is suggested to be part of the cleavage motif [64,

84, 169, 178, 201] could not be substituted by a

glycine, leucine, alanine, glutamine, threonine, or

phenylalanine residue without inhibition of process-

ing or processing at an incorrect cleavage site (Tanudjiet al., unpublished). −2Arg could not be exchanged by

other basic residues such as lysine or histidine without

partial or total inhibition of processing.

How important is the position of the proximal

arginine residue? There are plant precursors which

are processed although they lack a proximal arginine

residue and the presequence of tobacco pF1β does not

contain an arginine residue at position −2 but at posi-

tion −5. The −5Arg, in tobacco F1β could however be

substituted by a leucine or alanine residue without in-

hibition of processing. Results show that the proximal

arginine of alternative oxidase does not have to be atposition −2 but can also be at position −4 of the cleav-

age site in order for the precursor to interact correctly

with the active site of MPP. The optimal efficiency of

processing though, is achieved with a −2Arg.

Basic residues, distal to the cleavage site, have

been suggested to be important for processing in some

studies of mammalian precursors [87, 88, 142, 146,



328

149, 182] but not in others [76]. Studies of plant pre-

cursor proteins show that distal arginine residues to the

cleavage site of the tobacco F1β are not important for

the affinity of the presequence to MPP. The alternative

oxidase contains distal arginine residues at positions

−10, −30 and −35 and a lysine residue at position

−20. However, results indicate that −10 Arg is notimportant for processing whereas the other distal ba-

sic residues as distant of 30 and 35 residues from the

actual cleavage site, somewhat surprisingly, affected

processing of the soybean alternative oxidase with pu-

rified spinach MPP. However as a triple mutant of the

three distal positive residues was processed efficiently,

and a deletion mutant that had two of the distal positive

residues removed, this suggests that the distal residues

play a structural role essential for processing with the

entire presequence. Changing some of the residues

in single or double mutants can cause inhibition of

processing with purified MPP, further changes or dele-

tions relieves this inhibition. This indicates that these

residues do not have a role in the catalytic mechanism.

(Tanudji et al., unpublished).

It may be difficult to determine whether certain

amino acid positions within a given presequence are

particularly critical for cleavage function, or con-

versely, whether large segments of the presequence

contribute collectively to the functionality of the speci-

ficity. However, we can conclude from the muta-

tional analysis of the plant presequence that the most

proximal arginine is important, but not essential, for

processing of the soybean alternative oxidase (Tanudji

et al., unpublished) but not the tobacco F1β precur-sor (Sjöling et al., unpublished). In addition, basic

residues distant to the cleavage site, even those located

at the N-terminusof the presequence, are important for

processing of alternative oxidase. Surprisingly, dele-

tion of the amino acids flanking the scissile bond

of alternative oxidase, −1 serine and +1 glutamate,

inhibit but do not abolish processing.

Together with several other mutational analysis of

the alternative oxidase and the F1β precursor it can

be concluded that the secondary structure of the pre-

sequence is important, for processing, in some cases

together with distal and proximal arginine residues.

However, the conclusions are based on results of in

vitro studies. It is possible that additional factors may

influence the efficiency of processing in vivo.

Model of interaction of precursors with the MPP/bc1

complex

From our observations, and others, and with the

known structure of the bovine bc1 complex, we can

model the interaction and recognition mechanism of

a precursor protein with the plant MPP/bc1 complex.If the structure of the bovine core proteins, correlates

with the plant MPP/core protein structure, we can an-

ticipate that the presequence has to be flexible enough

to reach into the cavity of the core/MPP subunits to

the active site. Indeed, it has been shown that the pre-

sequence has to be flexible in order to be processed

[208]. We have also shown that the secondary structure

of the presequence is important for affinity to MPP

and thereby also for cleavage. A helix, proximal to

the cleavage site, would facilitate binding of the prese-

quence to MPP by helix/helix interaction. The amino

acids lining the wall of the cavity enclosed by the core

proteins in the bovine bc1 complex, are mostly hy-

drophilic and laced with negatively charged residues

[227], creating an environment which would be prone

to interact with positively charged presequences.

Two sets of basic residues enhance proteolysis in

several presequences, the proximal arginine within the

degenerate cleavage motif, and distant basic residues

relative to the cleavage site. The distance between the

proximal and distal residues varies between precur-

sors. Within this stretch a proline or glycine residue

may serve as a flexible linker and a helix breaker.

It would be possible for the basic charges to inter-

act with negative charges on MPP, and to present thescissile bond to the water on the metal in the active

site. A degenerate version of the potentially active

site of MPP, the inverted zinc binding motif, can be

found in the core 1 protein of the bovine bc1 complex,

Y91XXE94H95X 76E171. The residues Y91E94 and H95

are located on a helix facing the cavity and the core

protein 2 protein, not the matrix. Another helix, con-

taining the distal E, spans right above and across the

YEXXH helix [227]. This site, if we can correlate the

bovine structure to the spinach bc1 /MPP, may well be

the active site of MPP.

Models of evolution of the plant MPP

Why is plant mitochondrial MPP attached to the bc1

complex? The bifunctionality of this protein complex

has been suggested to reflect the co-evolution of two

enzymatic activities [26]. Bacteria have a bc1 com-

plex which lacks core proteins. The evolution of the



329

MPP and core subunits could have started with an an-

cestral protease which was hydrophilic and located in

the cytosol of bacteria, in correspondence to pitrilysin.

During endosymbiosis the processing peptidase might

have become attached to the membrane as it was ad-

vantageous for the function of the early MPP to be

located close to the protein import sites. Alternativelythe bc1 complex was dependent on new subunits ex-

posed to the matrix side for protection from proteases

that evolved in the matrix [15].

After gene duplication, a two component process-

ing peptidase evolved, corresponding to the present

situation in plant mitochondria. The detachment of

MPP from the bc1 complex in yeast, mammals and

partly N. crassa could reflect the necessity of inde-

pendent regulation of respiration and mitochondrial

import. The extra subunits of the bc1 complex could

have become indispensable for protection against pro-

teolytic degradation and assembly of the complex

[170] and therefore another gene duplication resulted

in the core proteins without catalytic activity and solu-

ble MPP in the matrix. The core proteins in yeast and

mammals would in this situation be evolutionary relics

of the processing peptidase [26].

What does not fit in this model is that the process-

ing activity of the mitochondrial processing peptidase

is independent of respiration [57] and therefore there

was no necessity for the MPP subunits to become de-

tached from the bc1 complex in yeast and mammals.

The evolution of the MPP subunits could have started

with a prokaryotic ancestor where a proteolytic ac-

tivity was integrated into the primitive bc1 complex,showing that the proteolytic activity could have been

present in the bc1 complex before the acquisition of

the core proteins.

Control of protein import into plant mitochondria

The control of protein import into mitochondria is an

area where studies with plants lead rather than are

derived from other organisms. This is a desirable sit-

uation as investigations in this area will not have to

overcome established paradigms that have been es-tablished in simpler organisms. Controlling the level

of protein within an organelle by controlling import

into that organelle would add another regulatory step

to the many characterised post-transcriptional control

steps known in plants [61]. It is generally believed

that the protein import into mitochondria is a consti-

tutive process. This impression arises from the fact

that the half-life of precursors in the cytoplasm is

generally very short [75, 189], deletion of an import

component in yeast is usually compensated by other

components or is lethal [141], and in vitro import stud-

ies usually have to use non-physiological inhibitors

to prevent import. That mitochondrial protein import

is constitutive is confounded by the fact that thesestudies are carried out with simple fungi grown under

ideal conditions, import under these circumstances is

probably constitutive and apparently rapid [141]. One

exception to the apparent universal constitutive import

ability of mitochondria in non-plant systems was seen

with two P450 precursor proteins from bovine. It was

clearly demonstrated in a number of reports that only

steroidogenic tissues could support the import of these

precursors, despite the fact that mitochondria from

other tissue were shown to be import competent for

other precursor proteins [126, 147]

In an attempt to develop an in vitro import sys-

tem from tobacco we have uncovered two possible

modes of regulation [45]. It has been puzzling, why

isolated respiratory competent tobacco mitochondria

only supported very low levels of import. Our stud-

ies demonstrated that it appeared that mitochondria

isolated from the dark phase of growth clearly im-

ported precursor proteins at much higher (∼4-fold)

levels than mitochondria isolated from the light period.

The failure of mitochondria isolated from the light

phase to import protein was apparently at the translo-

cation stage of import, as binding and processing of

the precursor protein was not effected by the time of

isolation. This indicated that some component of thetranslocation machinery was either absent or inacti-

vated during the light phase. Extending these studies

clearly showed that the inhibition was not directly due

to light as plants that were clonally propagated did

not excert this rhythmic variation in import. Clon-

ally propagated plants are continually wounded (or

stressed) and this appears to over rule any regula-

tion seen with the diurnal growth conditions. [43].

Likewise wounding of plants, by cutting off half the

leaves on the plant, allowing recovery for 1 to 2 weeks

showed not rhythmic pattern in import [43]. These

studies indicate that mitochondrial import in plants is a

regulated process and that this regulation is responsive

to outside signals.

Another level of control that has been uncovered

with plant mitochondria is developmental level. In

tobacco mitochondria it was found that the FAd sub-

unit of the ATP synthase could not be imported into

younger tobacco leaves (2 cm in diameter – 6–8 weeks



330

old) but could be imported into older tobacco leaves

(6 cm wide – 20 weeks old), in contrast the alternative

oxidase couldbe imported into both sets of leaves [45].

Developmental regulation of import has also been re-

ported with pea leaf mitochondria [49]. In this study

a dramatic decrease in import was observed between

day 6 and day 30 with pea leaves. This decline inimport was proposed to be due to a decline in the

level of mtHSP 70 as the amount of this protein also

decreased over a similar time period. However, the

decline in import was not strictly correlated with the

decline in mtHSP 70 levels, examination of the data

presented showed that even though import displayed

a five fold decrease from day 16 to day 30, that the

decrease in mtHSP 70 was less than two fold over the

same period. This indicates that other components or

factors effecting the activity of mtHSP 70 may also be

involved. A similar decrease in import ability has also

been reported for pea plastid import [42]. Therefore,

the decrease in mitochondrial import with pea mito-

chondria may represent an integrated cellular decrease

in organelle biogenesis with increasing maturity.

In a study of import during soybean cotyledon de-

velopment a 5-fold decline in import was observed

(Huang and Whelan, unpublished). Soybean cotyle-

dons are useful tissues for developmental studies as

they undergo a complete life cycle from germination

to senescence in approximately 20 days. Similar to the

study with pea mitochondria, a rise the amount of the

alternative oxidase was observed, but in contrast, we

detected a decline in components of the cytochrome

chain, in particular the Rieske FeS and cytochromeoxidase subunit II. The decline in these components

appeared to precede the decline in import. We propose

from these studies that the decrease in mitochondrial

import represents a general decline in mitochondrial

biogenesis. The fact that induction of the alternative

oxidase appears to be opposite to this general decline

can be explained by the fact that mRNA expression for

this subunit rises during this senescence period (Mc-

Cabe et al., unpublished) and this compensates for the

low import ability of mitochondria.

We believe that the recently uncovered mecha-

nisms of control of the protein import process repre-

sent the first studies and that the area is very fruitful for

further studies. Tissue, developmental and rhythmic

control of import are all possible avenues of regulation

of mitochondrial import. Additionally, it will be of in-

terest to uncover how the distribution of dual targeted

proteins is regulated in each organelle. One possibility

is that mitochondria (and plastids) are not constantly

importing proteins and that there exist factors that reg-

ulate the ability of organelles to import proteins; these

factors would determine the amount of proteins in an

organelle.

Concluding remarks

In the past ten years, impressive progress has been

made in characterisation the mitochondrial protein

transport machinery in plants. Although it appears

that the basic features of mitochondrial protein import

are conserved between all mitochondria [31], sev-

eral unique features have been unravelled in plants

in comparison to yeast and mammals. These features

include the following events and require further stud-

ies (i) structural characteristics of plant presequences;

(ii) the sorting phenomenon between mitochondria

and plastids, (iii) the involvement of cytosolic and

mitochondrial molecular chaperones, (iv) recognition

of the presequence by the import machinery and by

the processing peptidase, and (v) the rhythmic and

developmental regulation of the import process.

Statistical analysis of all available plant prese-

quences from the databases showed that plant prese-

quences are in average 7–9 amino acid residues longer

than in other species. They are enriched in especially

Ser, the basic residue Arg, and also Ala and Leu but

contain few Cys, His, Trp, Glu, Asp. There are typ-

ical MPP cleavage motifs with Arg in position −2

from the cleavage site R-X-A/S-T/S, or −3, R-X-X-

A/S-T/S, but the R-10 motif was not found in plantpresequences, indicating that MIP may not exist in

plant mitochondria.

In plants, the occurrence of the two endosymbiotic

organelles, mitochondria and chloroplasts requires

higher organellar specificity for protein import than in

non-plant sources. It is not known how this specificity

is maintained, but high specificity of import can be

achieved using specific targeting signals in combina-

tion with cytosolic factors. It has been also suggested

that specific cytosolic factors may direct mRNAs of

nuclear encoded organellar proteins into the vicinity

of a target organelle and increase specificity of import[120]. It is also possible that the receptor complexes

in plants contain additional components which would

contribute to a high specificity for organellar import in

plants. Next steps of investigations may thus involve

identification of novel or plant specific chaperones and

their role in the guidance of the newly synthesised

proteins to different intracellular biogenetic pathways



331

and also detailed characterisation of import receptor

complexes.

The cloning of several nuclear encoded mitochon-

drial proteins has allowed more detailed studies of

protein import into plants. However to date only the

import of one outer membrane protein (pTom 20) has

been studied. A few inner membrane proteins havebeen studied, but none in detail and little has been

done on assembly. Further studies to analyse different

import pathways will be of great interest. Of particu-

lar interest will be proteins that are nuclear encoded

in some plants but organelle encoded in others. An

example of this is seen with cytochrome oxidase II,

where it is nuclear encoded in some leguminous plants

[143]. Will this protein have novel features in import

or will it use the old import machinery. High order of

complexity has been observed in protein import into

thylakoids (Robinson et al., this issue).

Mitochondrial processing peptidase catalyses cleav-

age of several hundred mitochondrial precursor pro-

teins that are nuclear encoded, synthesised on cytoso-

lic polyribosomes and imported into mitochondria. In

contrast to non-plant sources where MPP is a matrix

enzyme, the plant mitochondrial MPP is localised in

the inner membrane and constitutes an integral part

of the bc1 complex of the respiratory chain. The bc1

complex in plants is thus bifunctional, being involved

both in respiration and in protein processing. How-

ever, despite the integration, the processing activity

is not dependent on the electron transport. Recent

studies unravelled also existence of a matrix located

processing activity in addition to the activity integratedinto the bc1 complex. It will be of interest to iso-

late and clone the gene of the protein catalysing this

activity and to understand basis for co-existence of

the membrane-bound and matrix-located processing

activities.

Although the length of the presequences ranges

from 8 to 121 residues (in all sources) and there

are no sequence similarities between the mitochon-

drial presequences and no consensus for the cleav-

age site, the presequence is cleaved off in a single

proteolytic process. Both the membrane-bound inte-

grated MPP/bc1

complex of plants and the soluble

mammalian MPP recognise similar higher-order struc-

tural elements upstream of the cleavage site that are

important for processing. The secondary structure

with flexibility and stabilising elements, hydrophobic-

ity and charge of the presequence seem to influence

the interaction with MPP. Prediction analysis of sec-

ondary structure shows that a helix structure followed

by an extended conformation is common in most

plant presequences, that could facilitate recognition

for processing by the unique plant MPP which is in-

tegrated into the bc1 complex of the respiratory chain.

As the structure for the mammalian bc1 complex is be-

coming unravelled, it will be interesting to model the

plant MPP/precursor interaction to understand recog-nition event and specificity of processing.

The regulation of protein import into mitochon-

dria in plants represents a new and potentially one

of the most far reaching aspect to emerge in recent

studies. The lower eukaryotes that have been exten-

sively studied are undifferentiated and in studies with

mammalian tissues, mature organs have been used.

In both these systems mitochondria are basically in a

constant environment, maintained by the growth con-

ditions with simple fungi and by integrated metabolic

control with higher animals. However, plants must be

able to respond rapidly to changing circumstances and

therefore it is not so surprising that they may have ad-

ditional levels of control not seen in other organisms.

To date both developmental and rhythmic control have

been uncovered. It is likely that tissue effects also exist

in plants. However if the mitochondrion can control

when it imports proteins it provides another means

to determine specificity. It also adds an extra level

of complexity to the communication that takes place

between the mitochondrion and the nucleus of which

there is little known in plants [185]. It will be of inter-

est to uncover as to how the mitochondrion ‘decides’

or is ‘told’ to import proteins. Will the different im-

port pathway be regulated differently and if so whatdistinguishes these pathway for such regulation. A

combination of different approaches will be necessary

to gain insights into this area.

In conclusion, over the last ten years, there has

been significant progress in the understanding of the

basic properties of the plant mitochondrial import

process. Several unique aspects of the process have

been discovered. Future studies using both in vitro

and in vivo techniques, combined with the informa-

tion from genome sequencing projects, will lead not

only to a greater understanding of this process in

plants but will also contribute to the understanding

of mitochondrial biogenesis in higher eukaryotes in

general.



332

Acknowledgements

This work was supported by grants from The Swedish

Natural Science Research Council (NFR) and The

Swedish Foundation for Strategic Research (SSF) to

EG and from The Australian Research Council to JW.

We are grateful to P. Dessi for Figure 2.

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7 - mitochondrial protein import in plants

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