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. bodies against mt-hsp 70. This suggests that in the A mechanism for unfolding can be proposed, how- mutant there may be a temperature-sensitive defi- elver, relying on further data obtained through the ciency in the dissociation of mt-hsp 70 from the analysis of the temperature-sensiti~-e.rscl-2 mutant. imported segments of the precursor polypeptides. The step of i2'I'P-dependent unfolding on the mito- Crosslinking experiments with a precursor protein chondrial surhce can be bypassed by artificially arrested in a membrane spanning fashion showed that unfolding precursors before import. In particular, this also in wild-type yeast precursors interact with mt-hsp was done with f~lsionproteins consisting ofaminoter- 70 before translocation is completed [Scherer et al. mina1 segments of the ATPase subunit 9 precursor and 1990). of complete mouse cytosolic dihydrofblate reductase As discussed above, import of most precursor pro- (DHFR). Upon unfolding (in 8 IVI urea followed by teins not only recluires ATP in the cytosol, but also rapid dilution), these SUS-DHFR precursors can be I\TP in the matrix (Hwang & Schatz 1989). 'T'ranslo- imported into isolated mitochondria without addition cation across the inner membrane becomes arrested of ,4'I'P, in contrast to import of the same precursors when the intramitochondrial ATP level is drastically out of reticulocyte lysate where they presumably are reduced by incubating isolated mitochondria with cornplexed with hsp 70. apyrase (an enzyme which degrades ATP and ADP) Import of these unfolded fusion proteins into iso- and by inhibition of the mitochondrial A'T'P-synthase. lated mitochondria from the .r.rcl-2 mutant was found ;\TP requirement in the matrix and the requirement to occur with almost the same kinetics as import into for functional mt-hsp 70 were found to be closely wild-type mitochondria, in sharp contrast to the correlated. situation with import from the reticulocyte lysate These findings are all compatible with a crucial role system (Kang et al. 1990). This obser\ration demon- of mt-hsp 70 in dri~-ingtlle translocation of the strated that the mutant rnitochondria mainly had precursors across the two mitochondrial membranes problems with importing precursors into the matrix by binding to the unfolded segments appearing on the when unfolding was a critical factor. Unfolding on the inside of the inner membrane (initially triggered by rnitochondrial surface, i.e. of those parts of a precursor AY), and thereby trapping the precursor chain step that are still present in the cytosolic compartment, is
I'hil. 'li.0171. R. Soc. Lorrd. R (1993') [ 1°1 I 358 \V. Neupert and N. Pfhnner Protezn targetzng znto mzlochondrza apparently fjcilitated by mitochondrial hsp 70 to- result \\-as found both in z~ie~oand after import of gether with matrix ATP. proteins into isolated mitochondria. Further hio- Hoxv could a component in the matrix then facili- chemical investigation then showed that hsp 60 is tate tlle unfolding of a protein on the other side of two directly in\-olved in the folding of individual polypep- membranes? As an answer to this question we propose tide chains. For its activity in facilitating folding, llsp that unfolding in the cytosol is essentially a sponta- 60 recluires A'TP hydrolysis. Upon reduction of the neous process. \Ye suggest that unfolcling occurs in iZTP level in the matrix, in the presence of a non- very small steps that require a very low amount of hydrolysahle A'TP analogue, ;\MP-PNP, or at energy. Each time such a small segment has assumed reduced temperature, a sloxv-down of fhlding in the an extended conformation it could slide in the 'trans- matrix was obser\-ed and the imported precursors location pore' and mt-hsp 70 on the other side I\-ould \\-ere largely found in association with the hsp 60 have tlle opportunity to bind and make the process complex (Ostermann el al. 1989). These results gatre eventually irreversible. Thereby, the ecluilibriu~ripro- the first experimental indication that folding of indi- cess of folding and unfolding could be turned into a vidual polypeptide chains in ixivo is mediated by a \sectorial reaction. chaperone. Experimental support for this pathway of' unfolding Ho~ve~~er,not only hsp 60 is in\vol\-ed in facilitating comes from the following obser\rations. Membrane folding in tlle matrix. \f:ith the .r.rcl-2 mutant it was spanning intermediates of cyt b2-1)HFR fusion pro- found that the refolding of urea-unfolded precursors teins (aminoterminal parts of tlle cytocllrome h2 imported at non-permissi~~etemperature was strongly precursor filsed to complete DHFK) can be accurnu- reduced. The precursors remained associated with mt- lated in the presence of rnethotrexate which stabilizes hsp 70 and were presumably not transferred to hsp 60 the DHF'R domain. Import occurs until the folded (Kang el al. 1990). It was therefore suggested that DHFR domain 'hits' the outer nlemhrane (Rassow et precursors ha\ve to be passed on from mt-hsp 70 to llsp al. 1989). Stabilization by the ligand clearly reduces 60 to become folded (Neupert et al. 1990; Langer el al. spontaneous unfolding strongly. 'The free energy of 1992). 'This \view has been questioned, however, and it stahilization is still rather low, being in the range of a has been claimed that pools of precursors not com- few kcal per mole. This is a \-er). small amount of plexed with either hsp 70 or hsp 60 exist jhfanning- energy compared with that needed for the ATP- Krieg et al. 1991). dependent release of the bound mt-hsps. ~Ilthoughit is not clear how many molecules of matrix ATP are (f)Hsp 70 and hsp 60 play important roles in required for importing an a\-erage-sized mitochondrial intramitochondrial sorting to the inner membrane matrix protein, the free energy needed for prel~enting and the intermembrane space unfolding !e.g. hv stabilization through a lieand) is ., \, , k, , almost certainly negligible compared to the free Some proteins that are transported to the matrix do energy of ,4'TP hydrolysis needed fbr recycling the mt- not remain there but are sorted to other suhmitochon- hsps. Also in agreement with the proposed unfolding drial compartments. Se\-era1 lines of elridence suggest mechanism is the fact that the translocation of the that molecular chaperones in the matrix are in\-ol\-ed folded DHFR domain (when methotrexate is remo\red in these processes. 11s an example, the precursor of the after accumulating transmembrane cyt b2-DHFR) is Rieske Fees protein (a component of complex 111 of much more dependent on temperature as compared to the respiratory chain anchored to the inner membrane the import of the urea-unfolded precursor (Eilers et al. and exposing a hydrophilic domain into the inter- 1988; Rassow el al. 1989). In summary, mt-hsp 70 in membrane space) requires both mt-hsp 70 and llsp 60 an indirect manner appears to play a decisive role in fhr becoming localized to its functional site. In the the unfolding of precursor proteins in the cytosol. sscl-2 mutant as well as in the m@ mutant its assembly was found to be impaired i Kang el al. 1990; Cheng et 01. 1989). A similar situation has been described for (e) Hsp 70 and hsp 60 in the matrix are involved in cytochrome bi! (a soluble component of the intermern- folding of polypeptide chains brane space) (Koll et al. 1992), although this pathway Mitochondria horn all sources analysed so far has recently been challenged (Click el al. 1992). contain the chaperonin hsp 60 which is structurally What is the role of the mitochondrial chaperones in and functionally closely related to GroEI, of prokaryo- these cases? The role of mt-hsp 70 appears to be to tic organisms [Reading et al. 1989; Ellis Pr Hemm- facilitate the entry of the polypeptides into the matrix ingsen 1989). This similarity appears to reflect the in the same way as it facilitates entry of matrix enclosymbiotic origin of mitochondria although hsp 60 c.omponents. A function of hsp 60 was proposed to lie of mitocllondria is encoded by a nuclear gene. I,ike in the protection of the intermediates in the matrix mt-hsp 70, hsp 60 is essential for the life of yeast cells against misfolding or aggregation befbre the ensuing i Cheng et al. 1989). export reaction [Koll et al. 1992). The absence of The analysis of the yeast mutant tniJl, which is folding by hsp 60 in these cases was thought to be the deficient in hsp 60 function in a temperature-sensitive result of the presence of sequences in the interme- manner, revealed an unimpeded import of precursors diates, the so-called sorting secluences, which would into the matrix, hut a lack of formation of acti~~e prelrent accluisition of a native folding state. It should matrix enzymes such as ornithine transcarbamoylase, be pointed out that this mode of sorting is likely to be F!-ArT'Pase or hsp 60 itself (Cheng el al. 1989). This the preferred one when import into the matrix is \,cry Prot~zntarg~ting into mztochondria 14'. Neupert and N. Pfbnner 359
triggering
translocation
ATP fold~ng / '+pi
Figure 1. Hypothetical model on the roles of molecular chaperones in mitochondrial protein import (modified after Neupert et al. 1990). C, carboxyltermiriu~of preproteiri; Ihl, iriner memhrarie; N, aminoterminus of preproteiri: OM, outer membrane. rapid. In the experiments described, in fact, import the overall reaction might then be provided by the was perfbrmed using urea-unfblded precursors whose transport into the intermembrane space, rather than import is 10-100-fold faster than import out of by mt-hsp 70 binding. This specialized pathway is, reticulocyte lysate. In the fbrmer case, export may not however, apparently not possible when longer seg- keep up in pace with import, and therefbre hsp 70 and ments of pre-cyt b2 precede the DHFR domain, hsp 60 come into play. possibly because the energy of the export reaction is It appears that in some cases both mt-hsp 70 and not sufficient to drive import. hsp 60 can be bypassed, in particular when export is fister than import. With certain precursor proteins, in particular with cyt D2-DHFR fusion proteins in which 3. CONCLUSIONS AND PERSPECTIVES the pre-cyt bz part is shorter than approximately 170 Our present knowledge and ideas about the roles of amino acid residues, a participation of mt-hsp 70 and molecular chaperones in mitochondrial protein sorting hsp 60 was not observed. In agreement with this, there are summarized in a working hypothesis which is was also no requirement for matrix ATP (unpublished depicted in figure 1. The overall reaction is divided results). It appears likely that under such conditions into three consecutive steps. import into the matrix and export into the iiitermem- brarle space are coupled events. The driving fbrce fbr 1. Triggering step. The hsp 70-complexed preproteins
Phll Tran~.R. Soc. Lund R (1993 I [ 103 ] 360 \V. Neupert and N. Pfanner P~ot~2ntatg~t~nt ~nto rnztociiondi~a interact with the receptor machinery in the outer the same pathway and the same components in an membrane and translocation of the aminoterminal identical manner. Alternative pathways, using targeting signal into the mitochondrial matrix takes unknown components or bypass pathways, should place. always be considered as being possible. This may be 2. Translocation step. Spontaneous unfolding on the particularly true because the fblding behaviour and cytosolic side, sliding of the extended precursor chain the binding capacity fbr molecular chaperones may in a 'translocation pore', and binding of mt-hsp 70 on not be unifbrm properties of all precursor proteins. the matrix side lead to gradual movement of the Future investigations will have to aim at a critical precursor into the matrix. ATP is required on both exami~~ationof our prcsent working hypothesis. They sides of' the two mitochondrial membranes. will have to detect a presumably large number OS 3. Folding step. The precursors are passed on fi-om additional chaperones and related components, they mt-hsp 70 to hsp 60 and become folded on the chaper- must dcfine the molecular mechanisms of' chaperone onin. IZTP is required for release of mt-hsp 70 and for action in mitochondria to provide a much more repeated cycles of binding and release ofthe polypep- precise picture, and they will have to search for tide on hsp 60 in the course of the folding process. additional functions of the known mitochondrial cha- perones.
It may be useful to look at the functions of \.VC arc gratrf~~lto Alexaridra \.Vrinzirrl for preparing thr mitochondrial molecular chaperones from an evolu- drawing. Studies from the authors' laboratorirs wrrc sup- portcd by tfie Ilentschc Forscfiurigs~en~eillscl~aft,tfic tionary angle. The prokaryotic origin of the mitochon- Hurnari I'rontier Scirncc Program and thc Fonds der dria is reflected in the amazing similarities of the Chrmischrn Industrie. molecular roles of hsp 60 and grol
ones: proteins essential fbr the biogenesis of some macro- \V. & Pfanner, X. 1990 Precursor proteins in transit molecular structures. Trends Biochem. Sci. 14, 339-342. through mitochondrial contact sites interact with hsp 70 F1)-nn, G.C., Chappell, 'I'.G. & Rothman, ,J.E. 1989 in the matrix. FEUS Lett. 277, 281-284. Peptide binding and release by proteins implicated as Phnner, N. & Neupert, \\. 1990 The mitochondrial catalysts of protein assembly. Scienrr, Wash. 245, 385-390. protein import apparatus. A. Rev. Biorhem. 59, 331-353. Georgopoulos, C., Ang, D., Liberek, K. & ZJ-licz, M. 1990 Phillips, G.,J. & Si1hav)-, 'I'.J. 1990 Heat-shock proteins Properties of the Escherichiu coli heat shock proteins and DnaK and GroEL facilitate export of LacZ hybrid their role in bacteriophage h growth. In Slres.c proteins in proteins in E. coli. ~Vuturr,Lond. 344, 882-884. biolog,y and medzcine (ed. 'l'. X4orimot0, A. 'l'issiers & Rasso~v,J. & Pfanner, X. 1991 hlitochondrial preproteins C. Georgopoulos), pp. 191-221. New York: Cold Spring en route from the outer membrane to the inner membrane Harbor Laborator). Press. are exposed to the intermembrane space. FEBS Lell. 293, Glick, B.S., Brandt, A., Cunningham, K., Muller, S., 85-88. Hallberg, R.L. & Schatz, G. 1992 Cytochromrs cl and hn Rasso~v,,J., Guiard, B., \Vienhues, U,, Herzog, \'., Hartl, are sorted to the intermembrane space of yeast mitochon- F.-U. & Neupert, \\. 1989 Translocation arrest by dria by a stop-transfer mechanism. C'ell 69, 809-822. reversible fblding of' a precursor protein imported into Ha~vlitschek,G., Schneider, H., Schmidt, B., Tropschug, mitochondria. A means to quantitate translocation con- hl., Hartl, F.-U. & Xeupert, \\. 1988 hlitochondrial tact sites. J. fill Biol. 109, 1421-1428. protein import: identification of processing peptidase and Rassow, ,J., Hartl, F.-U,, Guiard, B., Pfanner, N. & of PEP, a processing enhancing protein. Cell 53, 795 806. Neupert, \V. 1990 Po1)-peptides traverse the mitochon- Horwich, A. 1990 Protein import into mitochondria and drial envelope in an extended state. FERS Lett. 275, 190 peroxisomes. Curr. Opin. Crll Biol. 2, 625-633. 194. Hwang, S.T. & Schatz, G. 1989 'l'ranslocation of proteins Reading, D.S., Hallberg, R.L. & Llyers, A.hl. 1989 across the mitochondrial inner membrane, but not into Characterization of the )-east HSP 60 gene coding for a the outer membrane, requires nucleoside triphosphates in mitochondrial assemb1)- factor. Al'ulure, Land. 337, 655- the matrix. Proc. nutn. ilrud. Sci. LT.S.A. 86, 8432-8436. 659. Hwang, S.'I'., \Vachter, C. & Schatz, G. 1991 Protein Rothman, ,J.E. 1989 Polypeptidc chain binding proteins: import into the yeast mitochondrial matrix: a new catalysts of' protein fblding and related processes in cells. translocation intermediate between the two mitochon- C'ell 59, 591-601. drial membranes. J. biol. Chrm. 266, 21083-21089. Scherer, P.E., Krieg, U.C., Hwang, S.T., Vestweber, D. & Kang, P;J., Ostermann,,J., Schilling,,J., Neupert, \V., Craig, Schatz, G. 1990 A precursor protein partly translocated E.A. & Pfanner, X. 1990 Requirement for hsp 70 in the into yeast mitochondria is bound to a 70 kd mitochondrial mitochondrial matrix for translocation and folding of stress protein. EMBO J. 9, 431.5-4322. precursor proteins. Nuturr, Lond. 348, 137-143. Schleyer, M. & Neupert, \\. 198.5 Transport of proteins Koll, H., Guiard, B., Rasso~v,,J., Ostermann, ,J., Horwich, into mitochondria: translocational intermediates spanning A.L., Neupert, \V. & Hartl, F.-U. 1992 Antifblding contact sites between outer and inner membranes. Cell 43, activity of' hsp 60 couples protein import into the mito- 339-350. chondrial matrix ~vith export to the intermembrane \Vickner, \V., Driessen, A.J.14. & Hartl, F.-U. 1991 'l'he space. C'ell 68, 1163-1 175. enzymology of protein translocation across the Escherichiu Langer, 'l'., Lu, C., Echols, H., Flanagan, J., Ha)-er, M.K. coli plasma membrane. 11. Re!;. Riorhem. 60, 101-124. & Hartl, F.-U. 1992 Successive action of DnaK, DnaJ and GroEL along the pathwa)- of chaperone-mediated protein folding. ~Vuture,Lond. 356, 683-689. Discussion Llanning-Krieg, U.C., Scherer, P.E. & Schatz, G. 1991 Sequential action of mitochondrial chaperones in protein \V. J. \VELCH (Department of Medicine and Physiolop.y, import into the matrix. E,WBO J. 10, 3273-3280. University of C'alfornia, San Franscisco, U.S.A.). \$Thy do Llartin, J., Mahlke, K. & Pf'anner, N. 1991~Role of an proteins destined for the intermembrane mitochon- energized inner membrane in mitochondrial protein drial space have to interact with hsp 60? If hsp 60 import: AY drives the movement of presequences. J. biol. assists protein folding does this mean that the iron- Chem. 266, 18051-18057. Martin, ,J., Langer, T., Boteva, R., Schramel, A., Horwich, sulphur protein is active in the matrix? A.L. & Hartl, F.-U. 19916 Chaperonin-mediated pro- tein fblding at the surface of groEL through a 'molten \V. NEUPERT. I imagine that hsp 60 interacts with globule'-like intermediate. lklurr, Lond. 352, 36-42. most proteins that enter the matrix space and tries to hlurakami, H., Pain, D. & Blobel, G. 1988 70-kD heat assist its folding: it certainly does this even with shock-related protein is one of' at least two distinct fbreign proteins like DHF'R. But the protein may not c)-tosolic hctors stimulating protein import into mito- chondria. .J. Cell Biol. 107, 2051-2057. be capable of' being fblded because a signal sequence is Murakami, K. & hlori, M. 1990 Purified presecluence still present, or because of'the presence of' other fictors binding factor (PBF) fbrms an import-competent complex which prevent folding. So the extent of interaction with a purified mitochondrial precursor protein. EAfBO with hsp 60 will depend on these fkctors, and on the .I. 9, 32013208. kinetic situation, and may not be obligatory for Ncupcrt, \V., I-Iartl, I.'.-U,, Craig, E.A. 8r Pfanner, K. 1990 proteins destined fbr the intermembrane space. The How do polypeptides cross the mitochondrial mem- situation could resemble that suggested for the role of branes!' Cell 63, 447-4.50. GroEI, in protein transport in bacteria. Ostermann,,J., Horwich, A.L., Neupert, \V. & Hartl, F.-U. 1989 Protein fblding in mitochondria requires complex fbrmation with hsp 60 and A'I'P hydro1)-sis. ~Vuturr,Lond. 12'. J. ~VELCH.DO the authors think there may be a 341, 12.5-130. role for TCPl in binding to mitochondrial precursor Ostermann,,J., Voos, \V., Kang, P:J., Craig, E.A., Neupert, proteins in the cytosol?
Piiil. Tmrzj. K. Soc. Lorid. B (l9938 [ l05 1 362 It'. Neupert and N. PfBnner Protein targeting into mitochondria
12'. NEUPERT.I have no evidence on which to assess matrix space is binding to the matrix hsp 70, what is this possibility. the driving force for the transport fkom the matrix to the intermembrane space? This situation seems to A. BAKER (Department of Biochemistry, University of parallel the transport of proteins into the periplasmic Cambridge, U.K.). The authors suggest that the two space of' bacteria. different models fbr protein transport into the inter- membrane space can be reconciled by taking into It'. NEUPERT.It'e do not know the answer to this account kinetic factors. It is possible to change the interesting question. At the moment we can study only pathway of transport of, fbr example, cytochrome b2, the import and export steps at the same time, and by altering the rate of transport? what we require for such studies is a vesicle slstem which will transport from the matrix. There is of It:. NEUPERT.Kinetic fkctors will not be able to course ATP in the intermembrane space, but how reconcile the two models, as they are mechanistically proteins fold in this space is an open question. different, however, they may explain controversial interpretations of' experimental data. Since the two R. JAENICKE (Department of Biophysics and Physical models are not mutually exclusive it cannot be Biochemistry. Lrniversity of Regensburg, F.R.G.). How long excluded that different pathways might be favoured does the precursor protein on the cytosolic side of' the by different experimental conditions. mitochondrial membrane have to be to interact with hsp 70? A. BAKER.In the experiments with protein import into mitochondria fkom the hsp 60 mutant where the \V. NEUPERT.There is no systematic study of this authors fhund no mature form of the iron-sulphur aspect. Cytochrome c is a small mitochondrial protein protein, did they observe any mature protease-pro- and we have been unable to see any interaction tected form of cytochrome b2? between this protein and hsp 70; the transport of' this protein across the outer membrane into the intermem- \V. NEUPERT.There could be some present, but brane space does not require ATP on the cytosolic clearly less than in the control mitochondria. side.
11.-J. CETHING(Howa~d Hughes Medical Institute. Unizlersity of Texas, Dallas, Lr.S.A.). If, as the authors suggest, the driving force fbr protein import into the