Transport of Proteins Into Mitochondria Pfanner and Neupert 625

Transport o’f proteins into mitochondria

N. Pfanner and W. Neupert

Institute for Physiological Chemistry, The University of Munich, Munich, Federal Republic of Germany

Current Opinion in Ceil Biology 1989, 1:624-629

Introduction in vitro and in vivo. This development includes the characterization of properties of precursor proteins, the res- More, than 90% of mitochondrial proteins are encoded olution of distinct translocation intermediates on the im- by nuclear genes and are synthesized as precursor pro- port pathways and the identifkation of components of teins on cytosolic polysomes [ 11. The translocation of the protein import machinery. mitochondrial proteins from the cytosol to their functional destination in one of the four mitochondrial subcompartments (outer membrane, intermembrane space, Specific recognition of mitochondrial inner membrane and matrix; Fii. 1) involves a complex precursor proteins series of steps. The field of mitochondrial protein import has undergone very rapid development in recent Mitochondrial precursor proteins contain positively years. Most studies have used fungal mitochondria (the charged targeting sequences. In many cases, targeting sig- yeast saccbarom. c43?-a and NeuqDcwu crassa) nals are found in N-terminal peptide extensions of about

Fig. 1. Transport pathways of precursor proteins into mitochondria.

and translocation contact sites

Sorting of 0percursors

Outer membrane Matrix Inner membrane Int’ermembrane space

Abbreviations CIP-general insertion protein; hsp60-heat-shock protein of 60 kD; PEP-processing enhancing protein.

624 @ Current Science Ltd ISSN 0955a74 Transport of proteins into mitochondria Pfanner and Neupert 625

20-70 amino acid residues, termed presequences (Hurt tion of the ternary structure of precursor proteins (‘un- and van Loon, Trend Biochem Sci 1986,11:204-207) [ 21, folding’), e.g. by the introduction of point mutations, can which are cleaved off by specific peptidases inside the mi- increase the rates of import into mitochondria [ 91. tochondria. The main feature that mitochondrial targeting sequences have in common is the presence of positively Import of mitochondrial precursor proteins requires charged amino acid residues and the nearly complete ab- hydrolysis of ATP (Pfanner and Neupert, FEBS Left 1986, sence of negativeiy charged residues. In addition, target- 209:152-l 56; for a summary, see [lo] >. ATP is involved in ing sequences may form amphiphilic structures under the generation or maintenance of a translocation-compe- appropriate conditions [ 31. So far, no particular ammo tent (loosely folded) precursor conformation. In partic- acid sequence motif has been identitied in the target- ular, incompletely folded polypeptide chains require less ing peptides. This led to the suggestion that their sec- ATP for import than their more strongly foIded coun- ondary structure may have an important role in directing terparts [ 111. Translocation into the outer mitochondrial proteins into mitochondria. In several cases, the mature membrane of a chemically denatumted (unfolded) pre- (non-cleavable) regions of precursor proteins may con- cursor protein is completely independent of ATP [12]. tribute to the specifk high-athnity interaction of precur- Since the specilic import criteria, such as high-alfinity sors with binding sites on the mitochondria, possibly via binding to surface receptors, are fulfilled by the chemi- hydrophobic ‘assistant sequences’ [ 41. cally unfolded precursor [5], the role of ATP seems to be (directly or indirectly) related to modulation of the Proteinaceous receptors on the mitochondrial surface are conformation of precursors. Yeast mutants which are de- responsible for the specifk recognition and high-atfinity fective in a subset of stress proteins (‘heat-shock pro- binding of precursor proteins [5,6] (Pfanner and Neu- teins’) of the 70 kD class have been shown to be deficient pert, J Biol C&em 1987, 262:75287536). In the current in import of some mitochondrial proteins [13]. Seventy- working model, at least two distinct receptor proteins kilodalton stress proteins are generally thought to be in- are required for the binding of the various precursor volved in modification of the conformation of proteins in proteins (Fig. 2) [6]. High-affinity binding sites could an ATP-dependent manner [14]. In fact, Murakami et UI! be reconstituted into liposomes, providing an important [ 151 demonstrated that 70 kD stress proteins, and a fur- tool for the purifkation of receptor sites [5]. Antibod- ther cytosolic component, stimulate transport of a pre- ies which are directed against 45kD mitochondrial pro- cursor protein into mitochondria in vitro, The present teins inhibit import of precursor proteins, indicating that working model includes the participation of 70 kD stress a 45 kD protein could be involved in translocation of pre- proteins, and possibly other factors, in the generation or cursors (Ohba and Schatz, EMBO J 1987,6:2109-2115). maintenance of a transport-competent conformation of A detailed analysis of the role of the surface receptors precursor proteins. It seems unJikely that cytosolic factors gave rise to an explanation for a previously surprising re- also have a role in other steps of mitochondrial protein sult. Non-mitochondrial signals, such as chloroplast sig- import, such as targeting of precursors, since several pre- nal sequences, are able to direct proteins into mitochon- cursors can be imported in the absence of any cytosolic dria, albeit with a low efficiency (Hurt et al, EMBO J 1986, factor [ 5,101. 5:1343-1350). It was observed that precursor proteins The outer mitochondrial membrane contains apparently can bypass the proteinaceous surface receptors with a a common membrane insertion site used by all precursor very low efficiency, i.e. can enter the mitochondrial im- proteins studied so far with the exception of cytochrome port pathway at a later stage (PfaUer et al, J Biol C&m c (Fig. 2). This site, which is saturable with precursor pro- 1989, 264:34-39). This bypass transport appears to be teins, is termed the ‘general insertion protein’ (GIP) 161. the only pathway used by non-mitochondrial signals. The Insertion into the outer membrane seems to require a very low Import rates make it likely that bypass import high degree of unfolding of precursors and relatively high does not disturb the selectivity of mitochondtial protein levels of ATP (Pfanner et al, Cell 1987,49:81-23). After uptake and the unique mitochondrial protein composi- interaction with GIP, the precursors are inserted into the tion [7]. inner membrane. This step depends on the electrical potential across the inner membrane; the potential (negative inside) may exert an electrophoretic effect on the posi- Translocation of precursors into and across the tively charged targeting sequences (Pfanner and Neupert, mitochondrial membranes EMBO J 1985, 4:28192825) [3]. Butow and colleagues (Kellems et al, J Cell Bioll975, 65:1-14) proposed that Precursor proteins with a stable tertiary structure can- proteins are translocated at morphologically visible sites not be inserted into mitochondrial membranes (Eilers of close contact between mitochondrial outer and in- and Schatz, Nature 1986, 322:228-232). Precursors are ner membranes. The.experimental proof for transloca- at least partially unfolded before, or during, membrane tion of precursor proteins via contact sites was provided translocation. This can be clearly demonstrated by the by the demonstration of reversible trapping of precursors reversible accumulation of precursors spanning both mi- in contact sites (Schleyer and Neupert, 1985). Contact tochondrial membranes. The N-terminal portion of the sites are stable structures which can be enriched in sub- precursor enters the matrix space, whereas a (folded) C- mitochondrial vesicles (Schwaiger et a~!, J Cell Bioi 1987, terminal region remains in the cytosol (Schleyer and Ne- 105:235-246). Precursor proteins which are inserted into upert, cell 1985, 43:339-350) [8]. Moreover, destabiliza- contact sites are extracted from the membranes by pro- 626 Membranes

Cytosolic precursor proteins

ATP-dependent unfolding Pathway of cytochrome c: possible involvement of 70 kD stress proteins

lnsertion into the outer membrane Recognition by receptor proteins on the mitochondrial surface

Atachment of heme

Insertion into the outer Assembly into the outer membrane (general Intermembrane space membrane insertion protein)

Transport through contact sites between outer and inner membranes; electrical membrane potential for insertion into inner membrane

Proteolytic processing in the r\ Interaction with 60 kD proteins matrix (processing peptidase and w Assembly in the matrix c/ fhsp 60) in the matrix processing enhancing protein)

Re-translocation to inner membrane and intermembrane Assembly in inner space (‘conservative sorting); membrane and further proteolytic processing intermembrane space and attachment of prosthetic groups

Fig. 2 Mechanisms and steps of mitochondrial protein import.

tein denaturants such as urea or at alkaline pH [ 161. The Processing and folding df imported precursors contact sites can be saturated by membrane-spanning precursors [ 171. In summary, proteinaceous components The positively charged targeting sequence (presequence) seem to be of great importance for both the structure and is proteolytically cleaved off by the processing pepti- the function of contact sites. dase in the mitochondrial matrix. The processing pep- Transport of proteins into mitochondria Pfanner and Neupert 627 tidase was purified and was found to be a soluble pro- tors and with GIP and are then translocated through con- tein of about 57 kD [18]. A protein of about 52 kD tact sites into the matrix [ 11. which is mainly associated with the inner mitochondrial membrane greatly enhances the processing activity; it is Precursor proteins which are destined for the intermem- termed the ‘processing enhancing protein’ (PEP) [ 181. brane space or the inner membrane follow more elabo- However, the processing peptidase and PEP do not form rate sorting pathways (Figs. 1, 2). Most of them also use a detectable complex. PEP may bind precursor proteins surface receptors, GIP and contact sites for transport into protruding into the matrix space and then present them the matrix. After proteolytic processing and interaction to the processing peptidase. ‘lWo previously described with hsp60, the precursors are redirected into or across yeast mutants which are defective in maturation of mi- the inner membrane (Hattl et al, CeU 1986,47:939-951) tochondrial precursor proteins were found to be defi- [ 241. The mechanisms of retranslocation from the ma- cient in processing peptidase and PEP, respectively (Yaffe trix space into or across the inner mitochondrial mem- and Schatz, Proc Nat1 Acad Sci USA 1984,81:4819-4823) brane resemble the transport mechanisms in prokary- [ 19,201. The biochemical purification and characteriza- otes, the ancestors of mitochondria. This led to the fol- tion of these two components of the mitochondrial im- lowing model of intramitochondrial sorting (‘consetva- port machinery thus allowed the determination of the tive sorting’> [24]. The mitochondrial genes that otigi- functional defect in the two mutants. Processing pep- nally encoded mitochondrial proteins were transferred to tidase and PEP have significant homology in their pri- the nucleus and acquired a segment coding for a posi- mary sequences, suggesting that they may have originated tively charged presequence. The presequence directs the from a common ancestor [ 20,211. Several precursor pro- precursors through contact sites into the mitochondrial teins undergo a second proteolytic cleavage step either in matrix. After the first proteolytic cleavage, which results the mitochondrial matrix or in the intermembrane space in the removal of the positively charged targeting se- [ 11. The proteases which perform these processing steps quence, the precursor is released into its ancestral fold- have not yet been identied. ing and assembly pathway. Several precursors carry a second targeting signal in the C-terminal half of the prese- Recently, a new procedure for the selection of yeast quence. This relatively hydrophobic signal strongly re- mutants affected in mitochondrial protein import was sembles prokaryotic leader sequences which direct the worked out by testing the functional assembly of a export of prolouyotic proteins [I]. In fact, the second tar- nuclear-encoded matrix protein [20]. Several new mu- geting signal seems to direct translocation of precursors tants were found in addition to the two mutants de- across the inner mitochondrial membrane, and is cleaved scribed above. One of the mutants is defective in a consti- off at the intermembrane space side of the membrane. tutively expressed stress protein (‘heat-shock protein’) of Originally, it was proposed that the second (hydropho- about 60 kD (hsp60) [ 221. Hsp60 is homologous to the bic) signal sequence acts as a ‘stop-transfer sequence’ by chloroplast ribulose 1,5-bisphosphate carboxylase (Ru- preventing the further translocation of a precursor pro- bisco) subunit-binding protein and the Escbericbia coli tein into the matrix (van Loon and Schaa, EMBO J 1987, heat-shock protein groEL [23] (Hemmingsen et aA, Na- 6:2441-2448); the precursor protein would then reach its ture 1988,333:330-334). These three proteins are named location in the inner membrane or the intermembrane ‘chaperonins’, since they apparently ensure proper pro- space by lateral dilhtsion in the inner membrane. As dis- tein interaction during the assembly of oligomenc protein cussed above, this model does not apply to the sort- complexes. Hsp60 interacts with precursor proteins that ing pathways of precursor proteins studied so far. How- are transported into the mitochondtial matrix; this step is ever, the possibility that some precursors of integral inner a prerequisite for assembly of the precursors into protein membrane proteins may follow such a pathway cannot complexes and for the further transport of precursors be excluded. into, or across, the inner mitochondrial membrane (see below). Hsp60 may act by conferring a conformation on Cytochrome c, a protein of the intermembrane space, precursors which is required for assembty or intramito- uses an import pathway which is quite distinct from chondrial sorting [ 221. The action of a stress protein in the pathways of the other mitochondrial precursor pro- the mitochondrial matrix may represent a second ATP- teins. The precursor protein, apocytochrome c, can independent step in mitochondrial protein import, in ad- sert into the outer mitochondrial membrane without the dition to the ATP-dependent unfolding of cytosolic pre- aid of surface receptors or GIP 16,251. Cytochrome c cursors. heme lyase, a protein of the intermembrane space which is responsible for the covalent addition of heme to the apoprotein, seems to have a crucial role in the translo- lntramitochondrial sorting of precursors cation of cytochrome c into the intermembrane space. Mutants which are defective in cytochrome c heme lyase Precursor proteins destined for the outer mitochondrial are deficient in binding and import of cytochrome c membrane or the mitochondrial matrix follow relatively [26] (Dumont et al, EM30 J 1987, 63235-241). Apo- simple sorting pathways (Fig. 1,2). Outer membrane pro- cytochrome c may spontaneously insert into the outer teins interact with a’ specific receptor on the mitochon- membrane and binds with high atlinity, forming a com- drial surface, insert into the membrane, with the help plex which includes heme lyase. After addition of heme, of GIP, and then become assembled in the outer mem- the holoprotein is released into the intermembrane space brane. Matrix proteins also interact with specitic recep- [ 251. C-terminal portions of the precursor protein are ap- 620 Membranes

parently important for targeting of apocytqchrome c to targeting, whereas in many other presequences the N-terminal half car- mitochondria (Stuart et aA, EMBO J 1987,6:2131-2137). ries sufficient targeting information. Why are several (often very hydrophobic) proteins syn- 3. ROISE D, SCHAIZ G: Mitochondrial presequences. J Rio/ 0 &em 1988, 263:450+4511. thesized within mitochondria, although most mitochon- A minireview of structure and function of mitochondrial targeting se- drial proteins are encoded by nuclear genes? One pos- quences. sible explanation is that such hydrophobic proteins can- 4. PFANNER N, MOUER HK, HARM!ZY M4 NEUPERT W: Mitochon- not be translocated through the cytosol and the two 0 drill protein import: involvement of the mature part of mitochondrial membranes, or that they would not be a cleavable precursor protein in the binding to receptor able to assume the correct orientation and conforma- sites. EMBO J 1987, 6:34493454. tion for assembly when imported from outside the or- Hydrophobic ‘assistant-sequences’ in the mature part of some precursor proteins can - in addition to the effect of hydrophilic presequences ganelle. Nagkzy ef al [27] excluded these possibilities - increase the afi’mity for binding sites on the mitochondrial surface, using an elegant approach. They replaced the mitochon- and thereby increase the rate of import. drial gene for a membmne protein by an artificial nuclear 5. PFAUER R, NEUPERT W: High&inity binding sites involved gene that also contained a segment encoding a prese- 0 in the import of porin into mitochondria. EMBO J 1987, quence. The protein was successfiAly imported into mi- 6:2635-2642. tochondria and functionally assembled into a multi-sub- Preparation of large amounts of a pure precursor protein allows quan- unit complex. Hence, the most likely reason for the lack titation of high-attinity binding sites on the mitochondrial surface. The of transfer of the remaining mitochondrial genes to the binding sites are reconstituted into liposomes. Import of this (un- nucleus appears to be the divergence of the genetic code. folded) precursor is independent of cytosolic cofactors. 6. PFAL~IR R, STECER HF, RAYSOW J, PFANNER N, NEUPERT W: 0 Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common mem- Conclusions brane insertion site. J Cell Biol 1988. 107:24832490. Competition of tious precursor proteins for mitochondrial import Rapid progress in the field of mitochondrial protein im- sites reveals the existence of a common membrane insertion site, GIP, port led to the identiiication of several components of in the outer membrane which is used by precursors destined for dif ferent mitochondrial subcompartments. Distinct receptor sites precede the import machinery by biochemical and genetic ap- GIP in the pathway. proaches. The generation and characterization of defined transkxation intermediates on the import pathways of 7. PFANNER N, PFAUJZR R, NEUPERT W: How finicky is mitochon- l drial protein import? Trends B&&em Sci 1988, 13:165-167. precursor proteins opens the way for the identification Discusses mitochondrial protein import as a multi-step system. Some of of more components. The conformation of precursor the specific steps, such as binding to receptor sites, can be bypassed proteins is important for membrane translocation and with a very low efficiency. This provides an explanation for the surpris- assembly of proteins. Elucidation of the functional and ing result that non-mitochondrial signal sequences can direct proteins structural properties of mitochondrial precursor proteins into mitochondria. and of the transport components is not only essential 8. CHEN WJ, DOLIGW MG: The role of protein structure in for the understanding of mitochondrial biogenesis, but is a the mitochondrial import pathway. Unfolding of mitochon- also important for the unravelling of intracellular protein drially bound precursors is required for membrane translocation. J Biol ffxm 1987, 262:1560~15609. transport and biogenesis of cell organelles in general. A hybrid precursor protein with a folded C-terminal part is trapped in contact sites between both mitochondrial membranes, suggesting that unfolding on the mitochondrial surface is required for completion of Acknowledgements translocation. 9. VES~WTBER D, SCH.XR G: Point mutations destabilizing a We thank R.A Stuart for critical reading of the manuscript. a precursor protein enhance its post-translational import into mitochondria. EMBO J 1988, 7:1147-1152. The terrialy structure of a precursor protein is destabilized by site- directed mutagenesis, leading to an increased rate and efficiency of im- Annotated references and recommended pan. reading 10. EILERS M, SCHAR G: Protein unfolding and the energetics 00 of protein translocation across biological membranes. Cell a Of interest 1988, 52:481483. Minireview of the energetics of protein translocation and its relation to 00 Of outstanding interest unfolding of precursor proteins. Provides a very interesting comparison of the mechanisms used for translocation of proteins across various HARTL F-U. PFANNER N, NICHOLSON DW, NEUPERT W: Mi- 1. biological membranes. 0 tochondrial protein import. Biocbim BicpLys Acta 1989, 988:1-45. 11. VERNER K, SCHAIZ G: Import of an incompletely folded Very recent, comprehensive review of mimchondrial protein import. l precursor protein into isolated mitochondria requires an energized inner membrane, but no added ATP. EMBO J 2. HORW~CH AL, KALOUSEK F, FENTON WA FURTAK K, POLUXK 1987, 6:2449-2456. l RA, ROSENBERG LE: The omithine transcarbamylase leader Incompletely synthesized (nascent) chains of precursors are imported peptide directs mitochondrial import through both its mid- into mitochondtia. These loosely folded polypeptides require less ATP portion structure and ner positive charge. / Cell Biol 1987, for import than the corresponding complete (and folded) precursor 105664-678. proteins. A net positive charge is essential for the function of this presequence, as is the case for most mitochondrial targeting sequences. With this 12. PFANNER N, PFWR R, KIIENE R, 1’1’0 M, TROPSCHUG M, precursor protein, the midportion of the presequence is sufficient for 0 NEUPERT W: Role of ATP in mitochondrial protein import. Transport of proteins into mitochondria Pfanner and Neupert 629

Conformational alteration of a precursor protein can substi- Describes a new strategy for isolation of yeast mutants afTecting import tute for ATP requirement. J Biol C%em 1988, 263:4049-i051. and/or assembly of mitochondrial proteins. 1 of the mutants is deficient An artificially unfolded precursor of a mitochondrial outer membrane in processing peptidase. The primary sequence of processing peptidase protein is specifically imported into mitochondria. Its import is com- is homologous to that of PEP. pletely independent of ATP, suggesting that the ATP-dependence of protein import is related to modifications of the precursor conforma- 21. JENSEN RE, YAFFE MP: Import of proteins into yeast mito- tion. l chonti the nuclear MAS2 gene encodes a component of the processing protease that is homologous to the MASI- 13. DESHAIESRJ, KOCH BD, WERNER-W~HBURNE M, CRUC \x?, encoded subunit. EMBO J 1988, 738633872. l ~CHEKMAN R: A subfamily of stress proteins facilitates uans- Processing peptidase and PEP have homologous primary sequences. location of secretory and mitochondrial precursor polypep 22. tides. Nature 1988. 332~3W-804. CHENG MY, HAR’IL F-U, MARTLN J, PO~LOCK RA, KAL~USEK F, Yeast mutants which are depleted of a subset of 70 kD stress proteins l e NEUPERT W, HAUBERG EM, HAUBERG RL, NORWICH AL Mito- are deficient in import of precursor proteins into the endoplasmic retie. chondrial heat-shock protein hsp60 is essential for assem- ulum and mitochondria 70kD stress proteins thus may participate in bly of proteins imported into yeast mitochondria Natire both transport pathways. 1989, 337:62&24. Hsp60, which is located in the mitochondrial matrix, is required for 14. PELHAM H: Heat-shock proteins: coming in from the cold. assembly of precursor proteins. A yeast mutant deficient in hsp60 is also l Nature 1988, 3321776777. affected in the retmnslocation of precursor proteins from the matrix Brief discussion of the role of heat-shock proteins in intracellular pro- into or across the inner membrane 1241. tein t&Tic. 23. READINGDS, -ERG RI, MYEIS AM: (Letter to the Editor) 15. MURUW+U H. PAIN D, BL~BEL G: 70-kD heat shock-related l Characterization of the yeast HSP60 gene coding for a l e protein is one of at least two distinct cytosolic factors mitochondrial assembly factor. Nature 1989, 337z65%58. stimulating protein import into mitochondria. / Cefl Biol The primary sequence of yeast mitochondda hsp60 is homologous to 1988, 107:2051-2058. that of the E coli groEL protein and the Rubisco subunit-biding pro- Identilication of a 70 kD stress protein as a cytosolic factor stimulating tein of chloroplasrs. The 3 proteins (‘chaperonins’) facilitate assembly uanspon of a precursor protein into mitochondria in h-o. In addition, of protein complexes. evidence for a second cytosolic factor which acts synergistically with the 24. m F-U, OSTERMANN J, GU~ARD B, NEUPERT W: Succes- stress protein is presented. l e sive translocation into and out of the mitochondrial ma- 16. PFANNER N, HARTL F-U, GULARD B, NEUPERT W: Mitochondrial trix: targeting of proteins to the intermembrane space by l precursor proteins are imported through a hydrophilic a bipartite signal peptide. Cell 1987, 51:1027-1037. membrane environment. Eur / Bicdwn 1987, 169:289293. Several precursor proteins which are destined for the mitochondrial in- Mitochondrial precursor proteins which accumulate in contact sites in termembrane space are first completely imported into the matrix and a 2-membrane spanning fashion are extractable with urea or at alkaline are then redirected across the inner membrane. The principles of trans. pH (pH 11.5). Extraction by protein denaturants suggests that the pm location from the matrix across the inner membrane seem to be similar cursors were in a hydrophilic (proteinaceous) membrane environment. fo the mechanisms of protein export in prokaryotic cells, the ancestors of mitochondria. The sorting pathway via the matrix space is thus called 17. VESIWEBER D, S~HATZ G: A chime& mitochondrial precur- ‘conservative sorting’. l sor protein with internal disullide bridges blocks import of authentic precursors into mitochondria and allows quanti- 25. NICHOIS~N DW, HERGEWBERG C, NEUPERT W Role of cy- tation of import sites. J Cell Biol 1988, 107:2037-2044. l tochrome c heme lyase in the import of cytochrome c A precursor protein with a stably folded Ctenninal portion is irre- into mitochondria. J Bid c%em 1988, 263:19034-19042. versibly tmpped in mitcchondrial contact sites. In this way, impon of A tmnslocation intermediate is accumulated where the precursor of cy other precursor proteins is blocked and the number of translocation tochrome c spans the outer mitochondrial membrane and forms a tight conracf sites is assessed. complex with the cytochrome c heme lyase. After covalent attachment of heme, cytochrome c is completely translocated into the intermem- 18. HAwursc~~~ G, SCHNEIDER H, Sa+hwr B, TROPSCHUG M, brane space. l e HARTL F-U, NEUPERT W: Mitochondrial protein impon: identification of processing peptidase and of PEP, a processing 26. NARGANG FE, DRY&U ME, KWONG PI, NICHOLSON DW, NEUPERT enhancing protein. Cell 1988, 53~795-806. l W: A mutant of Neumspora crassa deficient in cy- Identification and functional characterization of the first 2 components tochrome c heme lyase activity cannot iniport cytochrome of the mitochondrial protein import machinery. The processing pepti- c into mitochondria. J Biol &em 1988, 263~9-394. dase which cleaves imported precursors is a soluble protein of the mi- Mitochondria from a A! crassa mutant which is defective in cytochrome tochondrial matrix, whereas PEP, which greatly stimulates proteolytic c heme lyase activity are deficient in binding and import of apo- processing, is largely associated with the inner membrane. cytochrome c, suggesting that cytochrome c heme lyase has an in-qor- rant role in the import pathway of cytochrome c 19. YANG M, JENSEN RE, YAFFE MP, OPPUGER W, SctLaz G: Im- 27. NAGEY P, FARREU LB, GEARING DP, NERO D, MELI~,ER S, l port of proteins into yeast mitochonti the purified ma- D!ZVENISH RJ: Assembly of functional proton-translocating trix processing protease contains two subunits which are l e encoded by the nuclear MASI and MAS2 genes. EMBO J ATF%.se complex in yeast mitochondria with cytoplasmi- 1988, 7:3857-3862. tally synthesized subunit 8, a polypeptide normalty en- 2 previously described yeast murants which are defective in maturation coded within the organelle. Proc NatI Acad Sci US4 1988, of mitochondrial precursor proteins are found to be deficient in pro- 85:2091-2095. cessing peptidase and PEP, respectively. A hydrophobic mitochondrial membrane protein which is normally syn thesized within mitochondria is expressed from an arti&al nuclear 20. POLLOCK RA, HARn F-U, CHENG MY, OSTERMANN J, HORW~CH gene which also codes for a presequence. The protein is imported into l 4 NEUPERT W: The processing peptidase of yeast mito- mitochondria and correctty assembled into a multi-subunit complex. chondrkz the two co-operating components MPP and PEP This elegant approach demonstrates that very hydrophobic proteins can are structurally related. EMBO J 1988, 7:34933500. also be imported from the cytosol.