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Roles of Molecular Chaperones in Protein Targeting to Mitochondria

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Roles of Molecular Chaperones in Protein Targeting to Mitochondria The Royal Society is collaborating with JSTOR to digitize, preserve, and extend access to Philosophical Transactions: Biological Sciences. ® www.jstor.org S56 \V. Neupert and N. Pfanner Profcin tacqeting into nzitochondria potential AY across the inner membrane is an abso- conformational state that difirs from that of their lute requirement for starting translocation into or mature forms, being more unfolded and exposing across the inner membranr:. It is 11ot entirely clear how hydrophobic 'sticky' patches. AY operates but a reasonable possihility is that the It is therefore generally assumed that del'rices must positively charged targeting sequences respond to AY exist in the cytosol to prevrnt misfolding and aggrega- in an c1ectrophori:tic manner (Martin ef al. 19910). tion of preproteins. X number of components haw This would explain why AY is only able (and heen implied in such a function. In particular, required) to trigger the translocation of the aminoter- cytosolic hsp 70s, termed Ssal-4p in yeast, ha~vebeen mina1 part (i.e. the presequence) of'the precursor, hut Sound to he important for the translocation of precur- tioes not play a rolc in the fbllowing translocation of sors into mitochondria iand also into the endoplasmic the main part of the molecule. reticulum). In mutant strains in \\-hich three of the After the precursor has partly or completely fbur SS,! genes xvere deleted, precursors of certain reached the matrix, the cleavable targeting sequence mitochondrial proteins accumulated in the cytosol is removed by a soluhle proccssing enzyme. '1~~0(Deshairs et al. 1988). Furthermore, experiments in components were found to be rccjuired for this reac- 2,iti.o similarly suggested that cytosolic hsp 70 stimu- tion, the mitochondrial processing peptidase (YIPP) lates protein import into mitochondria (Murakami et and the processing enhancing protein (PEP) (I-Tawlits- al. 1988). chek ef al. 1988). .\ fexv other cytosolic factors ha\-e also been de- 'l'he proteins then undergo fblding in the rnatrix to scribed which hind to precursors in the cytosol but lnonomers and oligomers. In those cases xvhere filrther which do not helong to the family of heat shock sorting takes place, in particular to the inner mem- proteins. Some of these factors halve heen implicated brane and the intermembrane space, reactions pre- in interacting specifically with the targeting sequences venting folding must occur (see beloxv). An importarlt (klurakami & Mori 1990). It rnight well be that a problern in this regard is in what conformational state di~verseset of proteins acts on various parts of the precursors are crossing the txvo mitochondrial mem- precursor proteins in the cytosol using difkrent strate- branes. It has become clear already some tirntx ago gies but halving a common aim, namely the preserlra- that unfolding of precursors is a prerequisite for tion of import-competence of precursors. In this transmernbrane 'transfer iEilers & Schatz 1986). llore respect rnitochondrial import may resernble protein recent studies have suggested that unfolding must be export frorn bacteria whcre hsp-type components, rather extensi\-e. It is a likely possihility that polypep- such as IhaK and GroEJ,, xvere found to interact tides tralversing the two membranes are in a f~llly xvith the presecretory proteins in the bacterial cytosol, evtcnded state. Measurements of the length of the hut also more specialized chaperones such as SecB segment OSa polypeptide chain that is spanning outer that do not belong to the group of heat shock proteins and inner ~nernbranesshow that as little as 50--60 (Wickner el al. 1991; Phillips Pr Silhavy 1990). amino acid residues are sufficient to span the two Protein import has a distinct rec~uirementfor ATP membranes oLrer a distance of about 15-20 nm (Kas- in the cytosol. This requirement is, at least in part, solv el al. 1990). This conclusion led to the suggestion due to the action of the heat shock proteins. In that translocation occurs in a manner in xvhich the particular, ATP hydrolysis seems to be required to polypeptide cllain is sliding through a proteinaceous release polypeptides from hsp 70s [ Rothman 1989). 'translocation pore' af'ter rnore or less complete unfold- This recluire~nentcan explain why hsp in\rol\,ement ing (Ncupert el al. 1990). and A'l~Phydrolysis are reactions that are usually found to parallel each other in experiments in rjitro. (6) A role of cytosolic hsp 70s in preserving precursor proteins in a transport-competent state (c) Hsp 70 in the mitochondrial matrix is essential for driving the translocation of precursors into The conibrmational state of precursor proteins in the matrix space the cytosol is usually difyerent frorn that of the nati1.e functional protein. It had been noticed already quite One of the eight llsp 70s of the yeast Saccharon~yc~s early in the study of mitochondrial protein irnport cei.evi.riaf, is localized within the mitochondria i Craig el that precursor proteins are extremely sensiti\-e to al. 1989). The product of this gene, called Ssclp or added proteases under conditions where the functional mitochondrial (mt-) hsp 70, performs essential fun(.- counterparts are resistant. Furthermore, a consistent tions in the cell as deletion of the SSCl gene results in observation is the strong tendency of precursors cell death (Craig et al. 1987). Mt-hsp 70 appears to be to\\-ards aggregation, both with preproteins synthe- a general constituent of mitochondria since it xvas not sized in cell-free systems and with preproteins only fbund in yeast, but also in humans, plants and rxpressed e.g. in E.rcherichia coli. In hct, precursor protozoa. proteins ~nadcin B. coli can be dissol\-ed in strongly :l terriperature-sensitive mutant afyectcd in SS(,,'/ denaturing media such as 8 M urea, then diluted and accumulated ~nitochondrialprecursor forms (Kang et imported into mitochondria; in most cases, ho\\-e\-er, al. 1990). iZfter a short time period following the shift such preparations are import-competent only for a frorn permissiLve to non-permissi~~etemperatures, thc short period of tirne, then aggregation takes place preproteins xvere found with the mitochondrial frac- jsurnmarized in Becker el al. 1992). These observations tion, yet they were not translocated into the mito- lrd to the conclusion that precursor proteins arc in a chondria sincc they \\-ere accessible to proteasc added Protezn targelzy znlo mztochondrza \.S. Neupert and N. Pfanner 357 to the isolated mitochondria. Detailed biochemical by step in the matrix space. A'I'P is required, accord- analysis of the mutant phenotype revealed a dual role ing to such a mechanism, to confer a conformation for this mt-hsp 70. It is in\-ollved in tlle translocation of competent for binding to mt-hsp 70, and ATP precursors across the two mitochondrial membranes, hydrolysis is required for the eventual release and and it plays an important role in the folding of recycling of mt-hsp 70. precursors inside tlle mitochondria. I\ salient feature of this proposed mechanism is the We will first discuss the experimental evidence for passage of the precursor into the matrix in an the role of mt hsp 70 in transmembrane movement of unfolded confbrmation. Only then would there be preproteins. Lt'hen precursors were imported into sunicient binding sites a,~ailable for dri~vingtransloca- mitochondria isolated from cells groxvn at a permissive tion in the direction of the matrix. We want to temperature and then exposed to a non-permissi~~e emphasize here that such a driving T-iahsp 70 binding temperature, processing to the mature-sized proteins may not necessarily be the only possible driving xvas obser~-edto almost the same degree as in identi- mechanism. In fact, we will discuss below that, with cally treated mitochondria from wild-type cells. How- some kinds of precursors of intermembrane space ever, when the mutant mitochondria were then proteins, the driving force may be pro~~idedby the probed as to whether they had imported these precur- export reaction from the matrix into the intermem- sors into the matrix by treatment with protease, the brane space. answer was no. 'The mutant mitochondria accumu- lated the precursors in a two-membrane spanning (d)Hsp 70 in the matrix has a role in facilitating fashion, i.e. the aminoterminus was able to enter the the unfolding of polypeptide chains on the matrix space but the mature parts of the precursors mitochondrial surface were still exposed to the extramitochondrial space. 'These findings were made with matrix proteins as well If transmembrane mo~-erntmtrequires an extensive as with proteins of the mitochondrial inner membrane unfolding of the polypeptide chain one may ask what and intermembrane space (Kang et al. 1990; Oster- the mechanisms are that fjcilitate unfolding on the mann et al. 1990). cytosolic side. As discussed abolre, precursors are These data suggested that mitochondrial precursors usually in a 'loosely fblded' conformation but these halve to interact with mt-hsp 70 to undergo transmem- conformations still contain abundant secondary and brane transfer, but that the onset fbr this requirement tertiary structure. lloreo\,er, it has been found that in was only after the precursors had completed the initial some cases domains of precursors assume a nati~ve translocation step that is triggered by the response of conformation when they are in the cytosol. Various the targeting sequences to AY across the inner speculations have been made on the existence of membrane. Direct support for such a mechanism unfolding components or 'unfoldases' that might inter- came from the demonstration that in the temperature- act lvith folded proteins in such a manner that the sensitive .rscl-2 mutant (but not in the wild-type) nati~ve folding state is disturbed. Sor far, no clear complexes between the precursors and mt-hsp 70 experimental e~-idencein h\-our of the existence of could be isolated by immunoprecipitation with anti- such unfoldases has been obtained.
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