The Biology of HEAT SHOCK PROTEINS and MOLECULAR CHAPERONES Edited by Richard I. Morimoto Northwestern University Alfred Tissieres University of Geneva Costa Georgopoulos University of Geneva COLD SPRING HARBOR LABORATORY PRESS 1994 Contents 1 Progress and Perspectives on the Biology of Heat Shock Proteins and Molecular Chaperones, 1 R . I . M o r i m o t o , A . T i s s i e r e s , a n d C. G e o r g o p o u l o s 2 Cytosolic hsp70s of Saccharomyces cerevisiae: Roles in Protein Synthesis, Protein Translocation, Proteolysis, and Regulation, 31 E . A . C r a i g , B . K . B a x t e r , J . Becker, J. H a l l a d a y , a n d T. Z i e g e l h o f f e r 3 Chaperoning Mitochondrial Biogenesis, 53 T. L a n g e r a n d W. N e u p e r t 4 Heat Shock Cognate Proteins and Polypeptide Translocation Across the Endoplasmic Reticulum Membrane, 85 J . L . B r o d s k y a n d R. Schekman 5 Structure, Function, and Regulation of the Endoplasmic Reticulum Chaperone, BiP, 111 M . - J . G e t h i n g S. B l o n d - E l g u i n d i , K . M o r i , a n d J.F. Sambrook y 6 Heat Shock 70-kD Proteins and Lysosomal Proteolysis, 137 J.F. D i c e , F. A g a r r a b e r e s , M . K i r v e n - B r o o k s , L.J. Terlecky andS.R. Terlecky f 7 Stress-70 Proteins and Their Interaction with Nucleotides, 153 D . B . M c K a y , S.M. W i l b a n k s , K . M . F l a h e r t y , J . - H . H a , M.C. O'Brien, a n d L . L . Shirvanee v vi Contents 8 Interactions of Vertebrate hsc70 and hsp70 with Unfolded Proteins and Peptides, 179 L . E . H i g h t o w e r , S.E. Sadis, and I.M. Takenaka 9 Properties of the Heat Shock Proteins of Escherichia coli the Autoregulation of the Heat Shock Response, 209 C. G e o r g o p o u l o s , K . L i b e r e k , M . Z y l i c z , a n d D . A n g and 10 Molecular Chaperone Functions of hsp70 and hsp60 in Protein Folding, 251 J. F r y d m a n a n d F . - U . H a r t l 11 The Basis of Recognition of Nonnative Structure by the Chaperone SecB, 285 L . L . R a n d a l l , T.B. T o p p i n g , a n d S.J.S. Hardy 12 The Structure, Function, and Genetics of the Chaperonin Containing TCP-1 (CCT) in Eukaryotic Cytosol, 299 K.R. Willison and H. Kubota 13 Modulation of Steroid Receptor Signal Transduction by Heat Shock Proteins, 313 S.P. Bohen and K.R. Yamamoto 14 Expression and Function of the Low-molecular-weight Heat Shock Proteins, 335 A.-P. Arrigo andJ. Landry 15 Structure and Regulation of Heat Shock Gene Promoters, 375 M . F e r n a n d e s , T. O ' B r i e n , a n d J . F . L i s 16 Structure and Regulation of Heat Shock Transcription Factor, 395 C. W u , J . C l o s , G. G i o r g i , R . I . H a r o u n , S.-J. K i m , S.K. R a b i n d r a n , J.T. Westwood, J . W i s n i e w s k i , a n d G. Y i m 17 Regulation of Heat Shock Gene Transcription by a Family of Heat Shock Factors, 417 R . I . M o r i m o t o , D . A . J u r i v i c h , P . E . K r o e g e r , S.K. M a t h u r , S.P. M u r p h y , A . N a k a i , K . S a r g e , K . A b r a v a y a , a n d L . T . Sistonen 18 Heat Shock Proteins and Stress Tolerance, 457 D . A . P a r s e l l a n d S. L i n d q u i s t Contents 19 Heat Shock Proteins as Antigens in Immunity against Infection and Seif, 495 S.H.E. Kaufmann and B. Schoel 20 Expression and Function of Stress Proteins in the Ischemic Heart, 533 I . J . B e n j a m i n a n d RS. W i l l i a m s 21 Postischemic Stress Response in Brain, 553 T.S. N o w a k , J r . a n d H . Ahe 22 Heat Shock Protein Gene Expression in Response to Physiologie Stress and Aging, 577 N . J . H o l b r o o k a n d R. U d e l s m a n Index, 595 vii 3 Chaperoning Mitochondrial Biogenesis Thomas Langer and Walter Neupert Institut für Physiologische Chemie München, Germany I. II. III. IV. V. I. Introduction Maintenance o f T r a n s l o c a t i o n C o m p e t e n c e in t h e C y t o s o l A. Conformation of Mitochondrial Proteins during Membrane Translocation B. Function of Cytosolic Chaperone Proteins C. Cytosolic Factors with Targeting Function for Mitochondria Protein T r a n s l o c a t i o n A c r o s s M i t o c h o n d r i a l M e m b r a n e s A. mt-hsp70-dependent Membrane Translocation B. Requirement for ATP in the Matrix C. Matrix ATP Requirement for Import of Intermembrane Space Proteins D. mt-hsp70-mediated Unfolding of Precursor Proteins E. mt-hsp70-independent Translocation Across the Outer Membrane of Mitochondria Folding and A s s e m b l y of M i t o c h o n d r i a l Proteins A. hsp60-dependent Assembly of Matrix-Iocalized and Inner Membrane Proteins B. Role of hsp60 for Sorting Proteins to the Intermembrane Space C. hsp60-mediated Folding of Monomeric Proteins in the Matrix D. Sequential Action of mt-hsp70 and hsp60 in the Mitochondrial Matrix E. Identification of the Mitochondrial DnaJ Homolog Mdj1 Perspectives INTRODUCTION The parallel development of powerful in vitro Systems and of genetic approaches has allowed considerable progress in understanding the mechanisms of protein transport into various cellular compartments. It is becoming ever more evident that transport processes across different cellular membranes are based on similar principles. Polypeptide chains appear to traverse lipid bilayers through proteinaeeous pores. Translocation requires a "translocation-competent," rather unfolded conformation. A s a consequence, proteins must be partially unfolded or kept in an unfolded conformation prior to the translocation event and must refold after Crossing the lipid membrane bilayer. In recent years, increasing evidence was obtained that both represent assisted processes. Molecular chaperones, in many cases originally identified as heat shock proteins, modulate the folding State of Polypeptide chains in different cellular compartments. T h e Biology of H e a t Shock Proteins and M o l e c u l a r Chaperones © 1 9 9 4 Colc Spring Harbor Uboratory Press 0-87969-427-0/94 $5 + .00 53 54 T. Langer and W. Neupert Mitochondria, which contain heat shock proteins of the hsp70 and hsp60 family, proved to be a useful model System to study the function of chaperone proteins in protein translocation and folding. Although mitochondria contain their own D N A and independent Systems for replication and protein synthesis, only a few subunits of the oxidative phosphorylation System and, in some organisms, components mediating splicing and translation of mitochondrial m R N A are encoded by the mitochondrial genome (Grivell 1989). About 95% of the total mass of mitochondrial proteins are encoded in the nucleus. They are synthesized on cytosolic polyribosomes, many of them as precursor molecules with amino-terminal presequences containing the targeting information. Import can occur posttranslationally followed by sorting to the various subcompartments of mitochondria, the outer and inner membranes, the intermembrane, and the matrix space. In recent years, an increasing number of components have been identified that are involved in the import and sorting of mitochondrial proteins (for reviews, see G l i c k and Schatz 1991; Segui-Real et al. 1992; Hannavy et al. 1993; Kiebler et al. 1993). In this chapter, we focus on the function of molecular chaperones in import and folding of mitochondrial proteins. In particular, we discuss their roles in maintaining a translocation-competent conformation of mitochondrial precursor molecules in the cytosol, in mediating the translocation process across mitochondrial membranes, and in the folding of matrix-localized proteins. II. MAINTENANCE OF T R A N S L O C A T I O N COMPETENCE IN THE C Y T O S O L A. C o n f o r m a t i o n of Mitochondrial Proteins d u r i n g Membrane T r a n s l o c a t i o n It is now generally agreed that proteins must attain a loosely folded conformation to traverse biological membranes, although with some organelles, in particular peroxisomes and glyoxysomes, the need for unfolding has not been proven. Studies of mitochondrial protein import provided direct experimental evidence for the requirement of a "translocation-competent" conformation of precursor proteins during the translocation process, which differs from the completely folded, native State: (1) Tight folding into a stable tertiary structure, e.g., induced by the presence of Substrate analogs or cofactors, was found to prevent the i m port of precursor proteins into mitochondria (Eilers and Schatz 1986; Chen and Douglas 1987; Rassow et al. 1989; Wienhues et al. 1991). Removal of the ligand restored import competence of the precursor protein. Conversely, destabilization of the native conformation by point Chaperoning Mitochondrial Biogenesis 55 mutations results in a more efficient import into mitochondria (Chen and Douglas 1988; Vestweber and Schatz 1988). (2) A nonnative conformation of precursor proteins during the translocation process is suggested by the identification of translocation intermediates spanning the inner and outer membranes (Schleyer and Neupert 1985). The two mitochondrial membranes form a barrier of about 10-12 nm as measured by electron microscopy. Using a set of fusion proteins consisting of aminoterminal parts of cytochrome b of various lengths and dihydrofolate reductase ( D H F R ) , Rassow et al. (1990) showed that about 50 amino acid residues are sufficient to span both mitochondrial membranes. This excludes that precursor proteins traverse membranes in their native conformation and suggests an extended or ß-sheet structure of the spanning portion of a Polypeptide chain, rather than an a-helical structure. 2 U p to now, physicochemical data have not been available that describe directly the conformation of translocation competent, mitochondrial precursor proteins. However, in view of the rapid collapse of proteins into a compact conformation after dilution from denaturant in vitro ( K i m and Baldwin 1990), a completely unfolded conformation of mitochondrial precursor proteins prior to membrane translocation seems to be very unlikely. Proteins were proposed to traverse membranes in a "moltenglobule -like conformation characterized by the presence of secondary structural elements and a flexible, disordered tertiary structure (Bychkova et al. 1988). A t this point, it should be noted that molecular chaperones, whose function in maintaining translocation competence is discussed in the following section, were found to stabilize unfolded proteins in a compact conformation without ordered tertiary structure (Martin et al. 1991b; Langer et al. 1992b). M B. Function of Cytosolic Chaperone Proteins A nonnative conformation of precursor proteins during membrane translocation implies that their folding must be modulated in the cytosol (Fig. 1). Precursor proteins to be transported across membranes could fold to the native State and become unfolded during membrane translocation (Pfanner et al. 1990; Skerjanc et al. 1990) or their folding is prevented in the cytosol. One obvious possibility would be that the amino-terminal presequence modulates the folding State of precursor molecules. After translocation, specific proteases within mitochondria cleave off the presequence, which would then allow folding to the native structure. However, the presequence is not sufficient to confer prolonged translocation competence. After dilution of mitochondrial precursor proteins from dena.urant into in vitro import assays, translocation competence is usual- 56 T. Langer and W. Neupert mt-hsp70 dependent import into m i t o c h o n d r i a F i g u r e 1 Possible mechanisms of maintenance of translocation competence of precursor proteins in the cytosol. ( S S A ) Ssalp/Ssa2p; ( Y D J ) Y d j l p ; ( X ) N E M sensitive factor; (mt-hsp70) mitochondrial hsp70. Chaperoning Mitochondrial Biogenesis 57 ly rapidly lost. In contrast, it was shown that precursor proteins are kept transport-competent for long periods in the cytosol, in many cases depending on the presence of A T P (Pfanner et al. 1987, 1990). Indeed, in recent years, several ATP-dependent cytosolic factors have been identified that stabilize various precursor proteins in the cytosol, preventing their folding or aggregation. A s discussed in a later section, however, (partial) folding of precursor proteins in the absence of these factors does not necessarily abolish translocation competence. Rather, unfolding in some cases can be promoted by the mitochondrial import machinery, in particular hsp70 (mt-hsp70), in the matrix (Fig. 1; see Section III.D). Studies in yeast revealed that molecular chaperones of the hsp70 family help to maintain a translocation-competent conformation of mitochondrial precursor proteins as well as proteins targeted to the endoplasmic reticulum, chloroplasts, and the nucleus (Chirico et al. 1988; Deshaies et al. 1988; Murakami et al. 1988; Waegemann et al. 1990; Dingwall and Laskey 1992). It is well established that hsp70 proteins interact with unfolded Polypeptide chains in an ATP-dependent manner. A m o n g the six cytosolic hsp70 proteins identified in yeast, evidence for a role in maintaining transport competence exists for S s a l p and Ssa2p. A yeast strain, in which the SSA1, SSA2, and SSA4 genes are deleted, could be rescued by expression of SSA1 from a galactose-regulated promoter (Deshaies et al. 1988). Genetic depletion of S s a l p resulted in the accumulation of precursor forms of the mitochondrial inner membrane protein F}ß and of a-factor in the cytosol, suggesting a common step in posttranslational protein transport across different membranes. The stimulating effect of Ssalp/Ssa2p on import of prepro-a-factor into m i crosomes was also demonstrated biochemically in in vitro transport Systems (Chirico et al. 1988). The identification of the temperature-sensitive yeast mutant mas3, which maps to the yeast heat shock factor (HSF), provides further evidence for a function of heat shock proteins in protein transport (Smith and Yaffe 1991). At the nonpermissive temperature, in the absence of an induction of SSA1, the rate of posttranslationally i m ported mitochondrial precursor proteins was decreased drastically. Interestingly, overexpression of S s a l p alone did not relieve this phenotype, indicating that additional heat shock proteins are functioning during eariy Steps of mitochondrial protein import (Smith and Yaffe 1991). Although a direct physical interaction of Ssalp/Ssa2p with prepro-afactor was recently demonstrated by coimmunoprecipitation (Chirico 1992), so far, no stable binary complexes were isolated between cytosolic hsp70 and mitochondrial precursor proteins. Therefore, a detailed description of the mode of action of hsp70 in the cytosol is still not available. Precursor proteins were described to be part of a 2 0 0 - 2 5 0 - k D 58 T. Langer and W. Neupert protein complex, which contains cytosolic hsp70 (Sheffield et al. 1990). ATP-dependent dissociation of the complex could be prevented by N ethylmaleimide ( N E M ) treatment of the cytosol. The N E M insensitivity of hsp70 proteins suggests the presence of an additional, NEM-sensitive subunit of the complex. Although this component has not yet been identified, eukaryotic homologs of the E s c h e r i c h i a c o l i heat shock proteins DnaJ and G r p E are attractive candidates. DnaJ and G r p E interact functionally with the E . c o l i hsp70 homolog D n a K (Liberek et al. 1991) and modulate its ATP-dependent interaction with unfolded Polypeptide chains (Zylicz et al. 1989; Liberek et al. 1991; Langer et al. 1992b). Indeed, a number of homologs of DnaJ were recently identified localized in various compartments of a eukaryotic cell (Kurihara and Silver 1992; Caplan et al. 1993). The Y D J 1 gene (also called M A S 5 ) was identified by Screening a yeast expression library with a polyclonal antiserum raised against a partially purified nuclear fraction (Caplan and Douglas 1991) and independently by Screening for yeast mutants displaying a defect in mitochondrial protein import (Atencio and Yaffe 1992). Subsequent biochemical analysis clearly demonstrated that Y d j l p is required for efficient posttranslational protein import into mitochondria (Caplan et al. 1992a). In temperature-sensitive yeast mutant strains at the nonpermissive temperature, precursor proteins of the a , ß, and y subunits of the F A T P a s e and of citrate synthase accumulate in the cytosol. The dependence of import on intact Y d j l p is obviously more strict at higher temperature. Only minor import defects were observed in a mas5 deletion mutant at 2 3 ° C , whereas cells were not viable at 3 7 ° C Interestingly, Y d j l p is farnesylated in vivo, which is essential for the function of the protein at high temperatures (Caplan et al. 1992b). Upon shift of the temperature to 3 7 ° C , the protein was partially relocalized to the membrane fraction dependent on the presence of the farnesyl lipid moicty. However, although enriched at the cytosolic side of the endoplasmic reticulum membrane, Y d j l p was not found in the outer membrane of mitochondria. A specific targeting function of the farnesyl group of Y d j l p during protein transport is therefore still speculative. r How does Y d j l p affect mitochondrial precursor proteins in the cytosol? The prokaryotic homolog DnaJ slightly stimulates the A T P a s e activity of D n a K (Liberek et al. 1991). This effect is far more pronounced in the presence of another heat shock protein, G r p E . Therefore, it is likely that Y d j l p exerts its effects in collaboration with hsp70 proteins in the cytosol. Indeed, purified Y d j l p functionally interacts with S s a l p , as it stimulates the ATPase activity of S s a l p up to ninefold (Cyr et al. 1992). Under these conditions, a permanently unfolded Polypeptide Chaperoning Mitochondrial Biogenesis 59 chain, carboxymethylated a-lactalbumin, was released from Ssalp/Ssa2p in vitro. It remains to be determined whether Y d j l p is indeed part of the described cytosolic complex of about 200-250 k D containing S s a l p / Ssa2p and mitochondrial precursor proteins (Sheffield et al. 1990). B e cause Y d j l p , as DnaJ, is found to be insensitive to N E M (D. C y r , pers. comm.; T . Langer, unpubl.), the complex should contain additonal components). Although not identified in the cytosol of eukaryotic cells up to now, a protein homologous to the E . c o l i G r p E protein is a likely candidate. C. C y t o s o l i c Factors with Targeting Function f o r M i t o c h o n d r i a In view of the involvement of Ssalp/Ssa2p and Y d j l p in the import of proteins into both mitochondria and endoplasmic reticulum, interaction of these molecular chaperones with the presequences, if it exists, apparently does not contribute to the specificity of targeting. However, chaperone proteins may stabilize precursor proteins in a conformation that allows interaction of the presequence with specific receptor proteins at the outer surface of mitochondria. Indeed, cytosolic targeting factors seem not to be absolutely required, as efficient import of a chemically pure preprotein into isolated yeast mitochondria was described to occur (Becker et al. 1992). The specific recognition of transport-competent precursor proteins by receptor proteins in the outer mitochondrial membrane is apparently sufficient for correct targeting in vitro. Nevertheless, cytosolic factors that bind specifically to mitochondrial presequences appear to exist, and several such factors have been identified in mammalian cells. A presequence-binding factor ( P B F ) was purified from rabbit reticulocyte lysate (Murakami and M o r i 1990). Whereas no interaction was observed with mature Ornithine transcarbamoyltransferase ( O T C ) , the precursor form was efficiently bound by P B F (Murakami et al. 1992). P B F is a homo-oligomeric protein of 50-kD subunits, with an S Q value of 5.5S. Depletion of rabbit reticulocyte lysate from P B F prevented i m port of O T C , aspartate aminotransferase, and malate dehydrogenase into mitochondria. Readdition of purified P B F fully restored import. In contrast, import of 3-oxoacyl-CoA thiolase, which lacks a cleavable presequence, did not depend on P B F . The mode of P B F action has not been understood up to now. Direct evidence for a chaperone-like role of P B F is so far lacking. It has been suggested that P B F might modulate the conformation of precursor proteins synergistically, with hsp70 proteins conferring additional mitochondrion-specific targeting information to the complex (Murakami et al. 1992). 2 w 60 T. Langer and W. Neupert Another cytosolic factor that stimulates mitochondrial protein import was isolated from rat liver (Ono and Tuboi 1988, 1990; Hachiya et al. 1993). This factor, termed mitochondrial-import-stimulating factor ( M S F ) , is composed of two subunits of 30 and 32 k D . In contrast to P B F , M S F exhibits strong A T P a s e activity in the presence of a transportincompetent precursor protein (Hachiya et al. 1993). A T P hydrolysis was reported to result in depolymerization of an in-vitro-synthesized mitochondrial precursor protein. Therefore, M S F may represent a novel chaperone protein specific for mitochondrial precursor proteins with a dual function: On the one hand, it may recognize presequences and confer translocation competence to precursor proteins; on the other hand, it may target precursor proteins to mitochondria. Interestingly, the two activities were affected differently by N E M treatment (Hachiya et al. 1993). Whereas no effect of the alkylating agent was observed on presequence binding and the ATPase activity of M S F , the stimulating effect of M S F on mitochondrial import was abolished, suggesting impairment of the release from M S F . N E M exhibited a similar effect on a cytosolic complex containing hsp70 and a mitochondrial precursor protein (Murakami et al. 1988; Sheffield et al. 1990). However, in contrast to P B F , Stimulation of import by M S F did not depend on cytosolic hsp70. The relative importance of M S F , P B F , or hsp70 for mitochondrial protein import in vivo remains to be determined. It might well be that a diverse set of factors interact with various parts of a precursor protein, resulting in stabilization of a transport-competent conformation and efficient targeting to mitochondria. III. PROTEIN T R A N S L O C A T I O N A C R O S S MITOCHONDRIAL M E M B R A N E S Translocation-competent precursor proteins are specifically recognized by receptor proteins at the outer surface of mitochondria, which are part of a protein complex in the outer membrane. This receptor complex consisting of at least six different proteins mediates binding and insertion into the translocation pore in the outer membrane of mitochondria (for review, see Kiebler et al. 1993). The targeting sequences are then thought to make contact with components of the inner membrane. Translocation of the presequence across the inner membrane into the matrix strictly depends on an energized inner membrane (Gasser et al. 1982; Schleyer and Neupert 1982). The electrical potential (Alp) may exert an electrophoretic effect on the positively charged presequence or influence the conformation of an inner membrane component in a manner such that translocation is triggered. This hypothesis is supported by the finding that differences in the positive Charge of presequences are reflected in a Chaperoning Mitochondrial Biogenesis 61 different sensitivity of import for the uncoupler carbonyl Cyanide mchlorophenylhydrazone ( C C C P ) (Martin et al. 1991a). Further translocation into the matrix does not require an energized inner membrane, but it does require the hydrolysis of A T P . The role of A T P for mitochondrial protein import was a matter of debate for a long time, mainly because A T P depletion experiments with isolated mitochondria were performed under various conditions, resulting in different A T P levels both outside and inside mitochondria. T w o ATP-dependent Steps of mitochondrial protein import are now well characterized. (1) In the cytosol, A T P is required to maintain a transport-competent conformation of precursor proteins, as discussed above. (2) The translocation of Polypeptide chains across the inner mitochondrial membrane is mediated by a matrix-localized hsp70 protein (mt-hsp70) in an ATP-dependent manner. A. mt-hsp70-dependent Membrane T r a n s l o c a t i o n First evidence for a function of mt-hsp70 in the translocation process was obtained upon characterization of the yeast mutant sscl-2, which contains a temperature-sensitive allele of the mt-hsp70 gene SSC1 (Kang et al. 1990; Ostermann et al. 1990). A t the nonpermissive temperature, precursor proteins of F}ß, hsp60, and S s c l p (mt-hsp70) itself accumulated in the cytosol in vivo. Consistently, import was impaired in in vitro Systems. The mutation in the SSC1 gene affected import of proteins of the inner membrane (e.g., the Rieske-Fe/S-protein and the A D P / A T P carrier), the intermembrane space (e.g., cytochrome C j ) , and the matrix (e.g., the ß subunit of the F A T P a s e ) . A t nonpermissive temperature in the sscl-2 mutant, these proteins accumulated at the surface of mitochondria, as assessed by their accessibility to externaily added protease. H o w ever, the amino-terminal presequences reached the matrix space and were cleaved off by the matrix-processing peptidase. Obviously, the accumulated translocation intermediates were spanning both mitochondrial membranes, indicating that mt-hsp70 acts already during membrane translocation. Indeed, a precursor protein partly translocated into the matrix could be cross-linked to mt-hsp70 (Scherer et al. 1990). In addition, electron microscopic studies revealed a localization of mt-hsp70 near the inner membrane (Carbajal et al. 1993). r On the basis of these results, a model for the translocation of proteins across mitochondrial membranes mediated by mt-hsp70 was proposed (Fig. 2) (Neupert et al. 1990; Neupert and Pfanner 1993). This model predicts cycles of binding of mt-hsp70 to an incoming precursor to provide the driving force for the translocation across the membrane. Spontaneous "breathing" of the Polypeptide on the outside would be suf- mt, hsp70J Receptor OM IM binding Presequence OM IM translocation ADP +R ATP 0 M , M Presequence Polypeptide stabilization translocation F i g u r e 2 Model of mt-hsp70-mediated membrane translocation of mitochondrial precursor proteins. A functional interaction of proteins homologous to E . c o l i DnaJ and GrpE is conceivable but remains to be demonstrated. (Mt-hsp70) Mitochondrial hsp70; (MPP) matrix processing peptidase; (OM) outer mitochondrial membrane; (IM) inner mitochondrial membrane. Chaperoning Mitochondrial Biogenesis 63 ficient to allow the passage of limited Segments of the precursor through the translocation pores in the outer and inner membranes. According to this view, binding of mt-hsp70 to incoming Segments of the precursor protein shifts the equilibrium of folded and unfolded State on the outside by trapping the unfolded precursor in a stepwise fashion on the t r a n s side of the two mitochondrial membranes. The model would also imply that breakdown of folded domains on the outside is a cooperative effect. After initial unfolding Steps, only little energy input is necessary, since then free energy stabilizing the folded conformation is no longer existing as a force preventing complete unfolding. The free energies that further stabilize a folded conformation upon binding of a ligand are usually in the ränge of a few kcal/mole, thus relatively small. Still, they are sufficient to block import efficiently (Eilers and Schatz 1986; Chen and Douglas 1987; Rassow et al. 1989; Wienhues et al. 1991). This would support the view that advantage is taken of the spontaneous reversible unfolding on the outside by the mt-hsp70-binding System inside. O b viously, the hsp70-binding/ATP hydrolysis System cannot work when spontaneous unfolding outside is strongly impaired by binding of a ligand. B. Requirement for ATP in the Matrix To test some predictions of this model, the energetics of membrane translocation was studied in more detail. mt-hsp70 mediates at least one i m portant ATP-dependent Step in the mitochondrial matrix during translocation. Therefore, the requirement of A T P in the matrix most likely reflects the function of mt-hsp70. A T P concentrations can be modulated in the matrix under various import conditions (Hwang and Schatz 1989; Stuart et al. 1994). In the absence of Substrates for the respiratory chain and by inhibition of the A T P synthase and the A D P / A T P carrier, A T P levels in the matrix can be decreased drastically in vitro. Reduction of the A T P concentration from normal levels of about 1.4 mM to 280 \m did not impair the translocation of matrix-localized proteins or proteins finally localized in the intermembrane Space (Stuart et al. 1994). However, at A T P concentrations of about 150 JIM, import of matrix-localized proteins like the ß subunit of the F A T P a s e or the S u 9 ( l - 6 9 ) - D H F R fusion protein was affected. The A T P available to mt-hsp70 under these conditions is extremely low since a considerable amount of total A T P in the matrix is bound to mitochondrial proteins, in particular to F r A T P a s e with affinities in the nanomolar ränge (Cross and Nalin 1981). Although the binding constant for A T P of mt-hsp70 has not been determined so far, it is expected to be in the micromolar ränge. D n a K , the E . c o l i hsp70 r 64 T. Langer and W. Neupert homolog, has an A T P - b i n d i n g constant of about 20 (LIM (Liberek et al. 1991). Therefore, most likely under conditions of extreme A T P depletion, mt-hsp70 in the matrix becomes inactive. Interestingly, at these extremely low matrix A T P levels, processing of precursor proteins was very inefficient and import-competent proteins accumulated at the surface of the mitochondria (Cyr et al. 1993). The Observation of inefficient processing of precursor proteins in the presence of Alp indicates that the presequence can reach the matrix. However, the membrane potential is not sufficient to translocate presequences across the inner membrane in a stable manner. In addition to Aip, A T P is required in the matrix. Precursor proteins, accumulated outside the inner membrane of mitochondria in the presence of Aip, but absence of matrix A T P , could be chased into the matrix by adding A T P . Most likely, the ATP-dependent interaction of mt-hsp70 with the incoming Polypeptide chain arrests the presequence on the matrix side of the inner membrane in a topology that allows cleavage by the matrix-processing peptidase (MPP) (Cyr et al. 1993). The Observation of only inefficient processing at low A T P concentrations suggests that already the binding of aminoterminal Segments of the precursor protein to mt-hsp70 requires the presence of A T P . Indeed, after import in ATP-depleted mitochondria, partly translocated S u 9 ( l - 6 9 ) - D H F R could only be coimmunoprecipitated with mt-hsp70 shortly after readdition of A T P (Manning-Krieg et al. 1991). Consistent results were obtained when two temperaturesensitive alleles of mt-hsp70, sscl-2 and sscl-3, were analyzed (Kang et al. 1990; Gambill et al. 1993). In the temperature-sensitive mutant sscl2, carrying a mutation in the putative peptide-binding domain, precursor proteins are bound to mt-hsp70 and processed efficiently at normal A T P levels. However, the release of bound Polypeptides is impaired. O n the other hand, in the temperature-sensitive mutant sscl-3, carrying a point mutation near the A T P - b i n d i n g site, which may prevent binding of A T P to mt-hsp70, binding and efficient processing were not observed. Taken together, these results indicate that ATP-dependent mt-hsp70 binding is sufficient to arrest the presequence in a stable manner on the matrix side of the inner membrane and allow efficient processing. C o m plete translocation of matrix-localized proteins into the matrix, however, requires several cycles of ATP-dependent binding and release from mthsp70. Even after unfolding of precursor proteins in vitro, import of matrix-targeted precursor proteins did not occur under conditions of extreme A T P depletion or at nonpermissive temperature in sscl-3 mutant mitochondria (Gambill et al. 1993; Stuart et al. 1994). Interestingly, under these conditions, efficient in vitro import of Polypeptides into the matrix was observed in the sscl-2 mutant, in which binding of precursor Chaperoning Mitochondrial Biogenesis 65 proteins to mt-hsp70 is still possible. This suggests that already the A T P dependent binding of (several) mt-hsp70 by itself to newly imported amino-terminal Segments of precursor proteins may be sufficient to drive the translocation, independent of the hydrolysis of A T P . C. Matrix ATP Requirement f o r Import of Intermembrane Space Proteins Import of several proteins localized to the intermembrane space was also found to depend on mt-hsp70 and matrix A T P . Whereas cytochrome b accumulates as a translocation intermediate spanning both mitochondrial membranes in the sscl-2 mutant under nonpermissive temperature, no processing was observed in the sscl-3 mutant or after A T P depletion of the matrix (Voos et al. 1993; Stuart et al. 1994). A stepwise reduction of A T P levels in the mitochondrial matrix during import revealed a lessstringent A T P requirement for sorting of proteins to the intermembrane Space compared to matrix-localized proteins. The hydrophobic part of the bipartite presequences in intermembrane space proteins that contain the sorting information relieves the requirement for the import for matrix ATP/mt-hsp70 (Voos et al. 1993; Stuart et al. 1994). A fusion protein containing the complete presequence of cytochrome b fused to mouse D H F R is transported to the intermembrane space even at very low matrix A T P levels or in the absence of functional mt-hsp70. In contrast, after deletion of the hydrophobic part of the presequence, which results in missorting of the otherwise identical precursor protein into the matrix, import is strictly dependent on the presence of A T P . Similar observations were made studying the import of cytochrome C j (Stuart et al. 1994). The efficient, mt-hsp70-independent processing of intermembrane space proteins in the matrix indicates that in this case, stable translocation of the presequence across the inner membrane is achieved by binding to another, not yet identified, protein that may interact with the hydrophobic part of the bipartite presequence. 2 2 The less-stringent dependence of intermembrane Space proteins on mt-hsp70 does not allow a differentiation between the models presently proposed for the sorting of intermembrane Space proteins, namely, the stop-transfer model and the conservative sorting model (Hartl and Neupert 1990; Glick et al. 1992). However, the efficient sorting at low matrix A T P levels of preproteins, loosely folded prior to import and destined to the intermembrane space, indicates that the precursor protein may be present in the matrix only with parts of the entire length at a given time. In frame of the conservative sorting model, this suggests that the Polypeptide chain is exported to the intermembrane space in a co- 66 T. Langer and W. Neupert translocational manner as proposed earlier ( K o l l et al. 1992). This mechanism could provide the energy for the movement of the Polypeptide chain from the matrix to the intermembrane space. D. mt-hsp70-mediated U n f o l d i n g of Precursor Proteins ATP-dependent binding to mt-hsp70 drives the vectorial movement of a precursor protein across mitochondrial membranes into the matrix. Several lines of evidence indicate that binding of mt-hsp70 to newly imported Segments of precursor proteins can indirectly promote the unfolding of domains at the outer surface of mitochondria, another key element of the model for its function in membrane translocation (Fig. 2): (1) The block of complete import of several precursors into sscl-2 mitochondria at nonpermissive temperature can be circumvented by urea denaturation of precursor proteins prior to import (Karig et al. 1990; G a m b i l l et al. 1993). (2) In contrast to various fusion proteins containing the presequence of cytochrome b , import and sorting of cytochrome b itself to the intermembrane space require matrix A T P and mt-hsp70 (Voos et al. 1993; Stuart et al. 1994). Cytochrome b , a lactate dehydrogenase, contains a tightly folded heme-binding domain (cytochrome-£> -like) followed by a flavin-containing domain. Upon protease treatment of the precursor, the cytochrome b domain is found to form a proteaseresistant fragment prior to import ( B . Glick; R. Stuart; both pers. comm.). A precursor protein, in which this domain was deleted, did not depend on matrix A T P and mt-hsp70 in its import (Stuart et al. 1994). Consistently, to reach the intermembrane space, fusion proteins containing aminoterminal parts of cytochrome b of various lengths and D H F R only required matrix A T P if the cytochrome b domain was intact. A s shown in sscl-2 mitochondria, urea denaturation of the precursor protein prior to import circumvented the necessity of A T P in the matrix for the translocation process. 2 2 2 5 5 2 5 A n unfolding reaction on the outside of the mitochondrion mediated by mt-hsp70 implies that folding of precursor proteins in the cytosol does not necessarily prevent efficient import (Fig. 1). Rather, import of folded (partially) proteins depends strictly on the action of mt-hsp70, which promotes unfolding outside. This is consistent with a mechanism by which unfolding at the mitochondrial surface occurs essentially in a spontaneous reaction. A s a consequence, cytosolic chaperones may not even be required to maintain a translocation-competent conformation of certain precursor proteins, one such example being cytochrome b . H o w ever, after stabilization of the folded structure by adding Substrate analogs or ligands, e.g., methotrexate for D H F R fusion proteins or heme 2 Chaperoning Mitochondrial Biogenesis 67 to a heme-binding domain, the energy provided by binding to mt-hsp70 (followed by ATP-dependent release) seems not to be sufficient to facilitate the unfolding. Under these conditions, translocation intermediates spanning across inner and outer membranes accumulate. From this scenario, it can be predicted that after removal of the ligand, e.g., methotrexate in the case of D H F R fusion proteins, the import of the translocation intermediates requires matrix A T P and mt-hsp70. Taken together, several key predictions of the current view of the mthsp70-mediated membrane translocation process (Fig. 2) received experimental Support. mt-hsp70 function could be studied either by A T P depletion of the matrix, preventing binding to mt-hsp70, or by characterizing temperature-sensitive mutants with a defect in binding ( s s c l - 3 ) or in release of Polypeptides ( s s c l - 2 ) . These approaches unraveled several functions of mt-hsp70: Presequences are stabilized in the matrix by A T P dependent binding to mt-hsp70. The vectorial movement of the complete Polypeptide chain across the two mitochondrial membranes requires several cycles of ATP-dependent binding and release from mt-hsp70. These interactions not only provide the energy for the translocation process itself, but can also promote unfolding of precursor proteins outside of mitochondria by shifting the equilibrium of folding to the unfolded State. E. mt-hsp70-independent T r a n s l o c a t i o n A c r o s s the Outer Membrane of M i t o c h o n d r i a Whereas the complete transport of Polypeptide chains across the mitochondrial inner membrane requires mt-hsp70 and the membrane potential, several precursor proteins can be translocated across the outer mitochondrial membrane in a manner independent of mt-hsp70. Cytochrome c, a soluble protein of the intermembrane Space, follows a quite exceptional import pathway (for review, see Stuart and Neupert 1990; L i l l et al. 1992b). Efficient import does not require receptor proteins at the surface of mitochondria or the hydrolysis of A T P . Attachment of the heme group in the intermembrane Space, catalyzed by cytochrome c heme lyase ( C C H L ) , and subsequent folding are thought to drive membrane translocation (Nicholson et al. 1988). C C H L , on the other hand, is transported via the receptor complex in the outer membrane into the intermembrane Space seemingly independent from an external energy source ( L i l l et al. 1992a). Neither A T P depletion nor destruction of the membrane potential across the inner membrane reduced the import efficiency. It is so far not clear how the energy is provided for the vectorial movement of C C H L across the lipid bilayer. Fold- 68 T. Langer and W. Neupert ing of C C H L or binding to a yet unidentified factor in the intermembrane space could drive the import reaction. Similarly, as in the matrix, a chaperone-like protein might be involved in these processes. However, the observed import of C C H L into isolated outer membrane vesicles argues against the requirement of a soluble factor in the intermembrane space (Mayer et al. 1993). Matrix-localized proteins cannot be imported into these vesicles, most likely because a driving force is missing that in intact mitochondria is provided by the simultaneous, mt-hsp70-dependent translocation across the inner membrane. IV. FOLDING A N D A S S E M B L Y OF MITOCHONDRIAL PROTEINS After membrane translocation, newly imported Polypeptides have to attain their native conformation at their site of function. In many cases, this seems to be an assisted process. A n increasing number of genes are being characterized whose functions are required for the assembly of protein complexes in the inner membrane, e.g., the F | F - A T P a s e , the ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase (for review, see Grivell 1989; Ackermann and Tzagoloff 1990; Luis et al. 1990; Buchwald et al. 1991). In many cases, the function of the products of these genes seems to be restricted to assisting assembly of a particular protein complex, i.e., they may function as "private" chaperones. In contrast, the molecular chaperone hsp60, localized in the matrix, was shown to mediate folding and assembly of many mitochondrial proteins (Cheng et al. 1989; Martin et al. 1992; Hallberg et al. 1993). 0 hsp60 belongs to a family of highly conserved proteins, termed chaperonins (cpn60) (Hemmingsen et al. 1988), that occur in prokaryotes (Hendrix 1979; Hohn et al. 1979) and eukaryotes, where it is present in mitochondria ( M c M u l l i n and Hallberg 1987, 1988; Jindal et al. 1989; Mizzen et al. 1989; Picketts et al. 1989) and in chloroplasts (Barraclough and Ellis 1980; Martel et al. 1990). hsp60 is encoded by an essential gene whose transcription is increased two- to threefold lipon temperature shift to 3 9 ° C (Reading et al. 1989). Under these conditions, the protein represents about 0.3% of total cell protein. A s other chaperonins, hsp60 is a homo-oligomeric protein composed of 14 subunits with a molecular mass of 60 k D . These subunits are arranged in two-stacked heptameric rings, thereby forming the characteristic barrel-like structure (Hutchinson et al. 1989). hsp60 exhibits an ATPase activity that is modulated by a cochaperonin (cpnlO) homologous to G r o E S in E . c o l i (Goloubinoff et al. 1989a). Although so far only identified in mammalian and plant mitochondria (Lubben et al. 1990; Hartman et al. 1992a,b), the ubiquitous occurrence of G r o E S homologs is very likely. Mitochondrial c p n l O Chaperoning Mitochondrial Biogenesis 69 proteins consist of seven identical 10-kD subunits that form a ring-like structure. A. hsp60-dependent A s s e m b l y of Matrix-localized and Inner Membrane Proteins The yeast H S P 6 0 gene was originally identified in the mutant m i f 4 that lacked enzymatic activity of imported mitochondrial proteins (Cheng et al. 1989). A t the same time, hsp60 was found in the yeast genome and its D N A sequence was determined (Johnson et al. 1989; Reading et al. 1989). Subsequent biochemical characterization of the temperaturesensitive mutant mif4 provided direct evidence for the involvement of hsp60 in the assembly of mitochondrial proteins (Cheng et al. 1989). Import of a number of precursor proteins localized in the matrix or the inner membrane was analyzed and found not to be affected at nonpermissive temperature in vivo. However, assembly of the ß subunit of the F j ATPase or of Ornithine transcarbamoylase and the maturation of the Rieske-Fe/S-protein were impaired. Under these conditions, a large number of matrix proteins, including Mif4p (hsp60), were found as aggregates in the membrane pellet after extraction of mitochondria. This points to a general role of hsp60 in the assembly of mitochondrial matrix proteins. Recently, these observations were further confirmed by genetic depletion of hsp60 (Hallberg et al. 1993). Yeast strains with a disrupted H S P 6 0 gene were rescued by expression of the wild-type gene from a galactose-inducible promoter. Growth of cells on glucose-containing medium resulted in depletion of hsp60. A s in m i / 4 mitochondria, proteins were imported normally but remained insoluble. Interestingly, as with other matrix proteins, hsp60 is required for its own assembly (Cheng et al. 1990; Hallberg et al. 1993). In addition, hsp60 also seems to be required for the assembly of some mitochondrially encoded proteins. In plant mitochondria, the newly synthesized a subunit of the F A T P a s e was found to be associated with hsp60 (Prasad et al. 1990). Taken together, these results demonstrate the requirement of the hsp60 complex for the biogenesis of mitochondrial matrix proteins. However, it was not possible on the basis of these studies to distinguish whether hsp60 affects folding or oligomerization of newly imported proteins. r B. Role of hsp60 for Sorting Proteins to the Intermembrane Space A function of hsp60 for sorting of proteins to the intermembrane Space is currently a matter of debate. A t nonpermissive temperature, accumula- 70 T. Langer and W. Neupert tion of the intermediate form of cytochrome b was observed in the mif4 strain in vivo (Cheng et al. 1989). Consistently, cytochrome b or hybrid proteins containing various amino-terminal parts of cytochrome b fused to D H F R were found in association with hsp60 in in vitro experiments ( K o l l et al. 1992). The association with hsp60 was taken as an additional evidence for the conservative sorting model which predicts that cytochrome b traverses the matrix on its sorting pathway to the intermembrane space. A s demonstrated in vitro using the purified E . c o l i hsp60 homolog G r o E L , A T P hydrolysis resulted in efficient release of the bound protein only if the hydrophobic part of the bipartite presequence of cytochrome b was deleted ( K o l l et al. 1992). This was interpretated to suggest an antifolding effect of hsp60 on intermembrane Space proteins with a bipartite presequence. The hydrophobic part of the presequence may promote a prolonged association with hsp60 that keeps the import intermediate in a conformation competent for re-export. 2 2 2 2 2 Recently, however, these results were challenged. A different phenotype was described for import of cytochrome b and cytochrome c^ into the intermembrane space of mitochondria isolated from the m i f 4 strain (Glick et al. 1992). In addition, both proteins were found to be imported with unchanged efficiency after genetic depletion of hsp60 (Hallberg et al. 1993). Although the latter result suggests that hsp60 may not be essential for correct sorting of cytochrome b and cytochrome C j into the intermembrane space, a kinetic effect of hsp60 was not excluded. hsp60 may stabilize intermediates in an export-competent conformation especially under conditions that favor import into the matrix over re-export into the intermembrane Space. This might explain the accumulation of the intermediate form of cytochrome b that was synthesized and i m ported into mitochondria after almost complete depletion of hsp60 (Hallberg et al. 1993). 2 2 2 C. hsp60-mediated Folding of M o n o m e r i c Proteins in t h e Matrix The demonstration of an impaired assembly of several newly imported proteins in the m i f 4 mutant strain at nonpermissive temperature raised the intriguing question of whether folding of monomeric proteins is mediated by molecular chaperones in vivo. A n assisted folding reaction might be required to cope with the high protein concentration in the mitochondrial matrix, which may be as high as 500 mg/ml (Schwerzmann et al. 1986), and thus favor aggregation of newly imported or synthesized Polypeptides. A hybrid protein containing D H F R fused to a mitochondrial targeting domain (amino acids 1-69 of subunit 9 of the A T P synthase; Chaperoning Mitochondrial Biogenesis 71 S u 9 [ l - 6 9 ] - D H F R ) was used to study folding of proteins within mitochondria (Ostermann et al. 1989). In the native conformation, D H F R exhibits an intrinsic protease resistance, allowing assessment of the folding State of the protein. In N e u r o s p o r a c r a s s a , folding of the D H F R was found to occur with a half-time of about 2 minutes (Ostermann et al. 1989). In contrast, spontaneous refolding of purified D H F R from denaturant in vitro takes place at a considerably faster rate (Touchette et ai. 1986). Together with the observed A T P dependence of the folding reaction in mitochondria, these results pointed to a role of hsp60 in mediating the folding of D H F R after import. Indeed, a stable complex of newly imported, unfolded D H F R with hsp60 was isolated from a mitochondrial matrix extract at reduced A T P levels (Ostermann et al. 1989). Addition of A T P resulted in folding of the D H F R in a protease-resistant conformation. Besides hsp60, an additional factor in the matrix was required for efficient folding, most likely a protein homologous to £ . c o l i G r o E S , in the meantime identified in mitochondria of various organisms (Lubben et al. 1990; Hartman et al. 1992a,b). Additional evidence for an hsp60 function in folding of D H F R was obtained by importing a fusion protein, p O T C - D H F R , into m i j 4 mitochondria in vivo (Martin et al. 1992). D H F R could only be extracted in a soluble, enzymatically active conformation at 23°C, whereas at the nonpermissive temperature, most of the protein was recovered in the membrane pellet. Therefore, despite the ability of D H F R to refold in vitro spontaneously after dilution from denaturant, in vivo folding is mediated by hsp60. This is also suggested by the slower kinetics of D H F R folding observed in vivo. These studies established the role of hsp60 in mediating the folding of newly imported, monomeric proteins. The general function of hsp60 is underlined by the Observation that under stress conditions (e.g., high temperature), hsp60 prevents the denaturation of a large number of preexisting mitochondrial proteins as well (Martin et al. 1992). After i m port into mitochondria in vivo, D H F R , a thermolabile protein, was inactivated at 3 7 ° C in the absence of functional hsp60 but was stabilized in an enzymatically active conformation in the presence of hsp60. A n A T P dependent association with hsp60 was only detected at high temperature, conditions that result in denaturation of D H F R . This suggests that under stress conditions in vivo, proteins are stabilized by ATP-dependent association with hsp60. Interestingly, in vivo, mt-hsp70 is not able to compensate for hsp60 in maintaining D H F R enzymatically active at 3 7 ° C . A detailed characterization of the folding activity of chaperonins, including mitochondrial hsp60, was performed by reconstitution of the folding reaction in vitro using purified components. These studies revealed principles of chaperonin action as reviewed elsewhere (Gething 72 T. Langer and W. Neupert and Sambrook 1992; Hendrick and Hartl 1993). Chaperonins of different origins, such as mitochondrial hsp60 and E . c o l i G r o E L , can Substitute for each other in in vitro folding assays, although with reduced efficiency (Goloubinoff et al. 1989b). This demonstrates the existence of a conserved, ATP-dependent mechanism of chaperonin action. The reduced efficiency of the folding reaction observed when using heterologous components may reflect the parallel evolution of the cochaperonin. D. Sequential A c t i o n of mt-hsp70 and hsp60 in the Mitochondrial Matrix In addition to hsp60, folding of newly imported proteins in the mitochondrial matrix requires functional mt-hsp70 (Fig. 3). A s discussed in an earlier section, mt-hsp70 promotes the translocation of Polypeptide chains across mitochondrial membranes by cycles c f ATP-dependent binding to the incoming protein. In the yeast mutant sscl-2 at the nonpermissive temperature, the block of import could be circumvented by urea denaturation of precursor proteins (Kang et al. 1990; Gambill et al. 1993). Under these conditions, a fusion protein containing D H F R ( S u 9 [ l - 6 9 ] - D H F R ) was imported completely into the matrix. However, D H F R remained bound to mt-hsp70 in an unfolded conformation as demonstrated by coimmunoprecipitation and by assessing its protease sensitivity after lysis of mitochondria. Obviously, protein folding requires the ATP-dependent release of newly imported proteins from mthsp70 and most likely transfer to hsp60. A sequential interaction of mthsp70 and hsp60 with newly imported matrix proteins was already suggested by the Observation that in contrast to mt-hsp70, functional inactivation of hsp60 did not affect the import reaction (Cheng et al. 1989). Indeed, upon import in vitro, the precursor of ß - M P P , a subunit of the dimeric matrix processing peptidase of yeast, could be coimmunoprecipitated with mt-hsp70 and hsp60 successively (Manning-Krieg et al. 1991). A T P hydrolysis promotes release from mt-hsp70 and binding to hsp60. In addition, newly imported hsp60 was found in a transient complex with mt-hsp70 prior to its assembly (Manning-Krieg et al. 1991). Interestingly, in hsp60-depleted mitochondria, newly imported hsp60 remained associated with mt-hsp70 even in the presence of A T P (Hallberg et al. 1993). Because of the lack of preexisting hsp60 oligomers, which are required for assembly (Cheng et al. 1990), hsp60 subunits remain bound to mt-hsp70. The Cooperation of mt-hsp70 and hsp60 in mediating folding of proteins localized in the mitochondrial matrix raises the intriguing question of how the ATP-dependent transfer of a Polypeptide chain from mt- hsp60 Membrane Folding translocation F i g u r e 3 Hypothetical model of the role of mitochondrial chaperone proteins in protein folding in the mitochondrial matrix. The direct functional interaction of M d j l p and of Y g e l p (see Section I V . D and E ) with mt-hsp70 in this process remains to be demonstrated. (Mt-hsp70) Mitochondrial hsp70; ( M D J ) mitochondrial DnaJ homolog M d j l p ; ( Y G E ) mitochondrial G r p E - h o m o l o g Y g e l p ; ( O M ) outer mitochondrial membrane; ( I M ) inner mitochondrial membrane. 74 T. Langer and W. Neupert hsp70 to hsp60 is regulated. Reconstitution experiments with purified components allowed further insights into the mechanism of chaperonemediated protein folding (Langer et al. 1992b). The homologous proteins from E . c o l i , D n a K and G r o E L , were used in these studies that share 58% ( D n a K and S s c l ) and 54% ( G r o E L and hsp60) sequence identity with their mitochondrial counterparts (Craig et al. 1989; Reading et al. 1989). The transfer of a protein from D n a K to G r o E L was found to be tightly regulated. The ATP-dependent interaction of an unfolded Polypeptide chain with D n a K is modulated by two other heat shock proteins of E . c o l i , DnaJ and G r p E (Liberek et al. 1991; Langer et al. 1992b). Binding of DnaJ, which can act as a molecular chaperone on its own, increases the affinity of D n a K for an unfolded protein (Zylicz et al. 1989; Wickner et al. 1991; Langer et al. 1992b). A complex between D n a K and DnaJ is formed that is stabilized by A T P hydrolysis by DnaK. G r p E mediates the A D P release from D n a K , resulting in a decreased Substrate affinity of D n a K . Under these conditions, an efficient transfer of the protein to G r o E L is observed. Subsequently, folding of the Polypeptide chain occurs in association with G r o E L in an ATP-dependent manner, most likely within the central cavity of the G r o E L cylinder (Martin et al. 1991b; Langer et al. 1992a; Braig et al. 1993). The sequential interaction of the molecular chaperones, D n a K and G r o E L , seems to be directed by their binding specificity (Langer et al. 1992b). Whereas D n a K , like various eukaryotic hsp70 proteins (Palleros et al. 1991), exhibits high Substrate affinity for Polypeptides that are in an unfolded conformation lacking secondary structures, a Polypeptide chain in the process of folding to its native State is stabilized by G r o E L in a collapsed State characterized by a disordered tertiary structure (Martin et al. 1991b). The successive interaction of D n a K and G r o E L with a Polypeptide chain during its folding in vitro may mimic the Situation prevailing in mitochondria. Participation in mitochondria of proteins with a function similar to that of E . c o l i DnaJ and G r p E is an attractive possibility. Indeed, a protein homologous to E . c o l i G r p E was recently identified ( Y g e l p ; E . Craig, pers. comm.) that was localized to the mitochondrial matrix. In agreement with the predicted function, the protein is encoded by an essential gene (E. Craig, pers. comm.). On the other hand, a general importance of DnaK-DnaJ-like interactions in eukaryotes is suggested by the identification of proteins homologous to E . c o l i DnaJ in various compartments of a eukaryotic cell; part of them has already been shown to interact functionally with hsp70 proteins (Kurihara and Silver 1992; Caplan et al. 1993). In Saccharomyces c e r e v i s i a e , SCJ1 (37% sequence identity to DnaJ) was identified as a gene whose overexpression results Chaperoning Mitochondrial Biogenesis 75 in missorting of a nucleus-targeted cytochrome Cj fusion protein to mitochondria (Blumberg and Silver 1991). However, a mitochondrial localization of S c j l p by cellular subfractionation has not been demonstrated. E. Identification of the Mitochondrial DnaJ H o m o l o g Mdj1 Recently, during D N A sequencing of an S. c e r e v i s i a e X clone library, another gene was identified that turned out to encode a mitochondrial DnaJ homolog and was therefore termed M d j l p (mitochondrial DnaJ) ( N . Rowley et al., in prep.). The M D J 1 gene exhibits striking similarity with already known DnaJ homologs. The gene encodes a protein of 511 residues that is 33% identical to E . c o l i DnaJ over the entire length. Moreover, the characteristic sequence motifs found in DnaJ homologs are also present in the M D J 1 gene. The "J region" of M d j l p is 50% identical to that in E . c o l i DnaJ and 54% identical to that in Y d j l p , which is located in the cytosol of S. c e r e v i s i a e . In addition, M D J 1 contains a glycine-rich region as well as a four times repeated cysteine-containing motif in the central part of M d j l p , both motifs being characteristic for members of the DnaJ family. In contrast to other known DnaJ homologs, an amino-terminal extension is found in M d j l p that is rieh in basic amino acids, a characteristic feature of mitochondrial presequences. Indeed, M d j l p is synthesized as a larger presursor protein and imported into isolated mitochondria followed by cleavage of the presequence. The protein was localized to the mitochondrial matrix, more precisely to the inner side of the inner membrane. To analyze the function of M d j l p within mitochondria, a gene disruption was carried out ( A m d j l ) . A s with other DnaJ homologs, M d j l p is not essential for viability. Disruption of the M D J I gene resulted in a p e t i t e phenotype in yeast. Whereas normal growth on fermentable carbon sources at 3 0 ° C was observed, cells were inviable at 3 7 ° C and unable to grow on nonfermentable carbon sources at any temperature. Growth at 3 7 ° C could be restored by transformation of the disruptant strain with the complete M D J 1 gene. Interestingly, M D J 1 is required for the maintenance of mitochondrial D N A . N o mitochondrial D N A was found in the disruptant strain. In view of the known funetions of other DnaJ homologs, in particular their functional interaction with hsp70 proteins, an impaired protein import or folding may aecount for this effect. Alternatively, the observed p ° phenotype may reflect a function of M d j l p in mitochondrial D N A replication or in translation, which is required for maintaining mitochondrial D N A . T o obtain further evidence for the role of M d j l p in mitochondrial biogenesis, protein import and folding within mitochondria were studied. 76 T. Langer and W. Neupert In contrast to an inactivation of mt-hsp70, disruption of M D J 1 did not affect protein import into different mitochondrial subcompartments. Matrix-localized proteins (e.g., ß subunit of the F j - A T P a s e ) , proteins located in the intermembrane space (e.g., cytochrome b ) , as well as proteins of the inner (e.g., A D P / A T P - c a r r i e r ) and outer membranes (e.g., M O M 3 8 ) were imported in the absence of M d j l p with the same efficiencies and kinetics as those observed in wild type. In contrast, M d j l p seems to participate in the folding of both newly imported and preexisting proteins within mitochondria. After import of D H F R fusion proteins into mitochondria, protease-resistant folded D H F R was formed even in the absence of M d j l p , however, with reduced efficiency. In the A m d j l strain, insoluble D H F R was in the pellet fraction after low-speed centrifugation, most likely representing aggregated protein. This indicates a role of M d j l p in folding of newly imported proteins and, in addition, in folding of preexisting proteins. Completely imported D H F R exhibited a decreased heat stability at 3 7 ° C in A m d j l , suggesting a role of M d j l p in stabilization of preexisting mitochondrial proteins against heat denaturation. 2 Taken together, these results demonstrate the importance of M d j l p for the formation of respiratory-competent mitochondria. Further experiments are required to demonstrate a functional interaction with S s c l p in mediating import and folding of proteins. Such a Cooperation would also point to a protein homologous to E . c o l i G r p E recently identified in yeast (as discussed above). It may seem surprising that deletion of M D J 1 affects folding of mitochondrial proteins, but not membrane translocation, in view of the participation mt-hsp70 in both processes. Studies of the function of E . c o l i D n a K demonstrated that D n a K binds extended Polypeptide chains with high affinity, whereas a stable complex with compact folding intermediates can only be detected in the presence of DnaJ (Langer et al. 1992b). Therefore, different conformational states of precursor proteins during membrane translocation and during subsequent folding may account for the different effects of M D J 1 deletion. The observed requirement of M d j l p for maintenance of mitochondrial D N A may suggest a role of M d j l p in mitochondrial D N A replication or protein synthesis, comparable to DnaJ in the replication of viral D N A in E . c o l i (Georgopoulos et al. 1990) or S i s l p in initiation of translation in S. c e r e v i s i a e (Zhong and Arndt, 1993). V. PERSPECTIVES Molecular chaperones are known to fulfill essential functions during biogenesis of mitochondria. Although general functions are recognized, Chaperoning Mitochondrial Biogenesis 77 in many cases, a detailed analysis of the mode of action of the various chaperones is still awaited. This holds particularly true for their role in maintaining a translocation-competent conformation in the cytosol. Questions addressing the composition of cytosolic complexes that contain mitochondrial precursor proteins, the coordination of various chaperone proteins in the cytosol, or the interplay with targeting factors specific for mitochondrial presequences need to be answered. Future studies should also allow further insights into the function of chaperone proteins within mitochondria. Although a sequential action of mt-hsp70 and hsp60 has been described, the general importance of this pathway is still a matter of debate. The analysis of M d j l p mutants may provide further clues as to how the Cooperation is regulated. Moreover, mitochondrial chaperone proteins may also participate in processes other than protein import and folding, such as D N A replication, D N A recombination, protein synthesis, and degradation. Again, mitochondria may turn out to represent a useful model System to discover novel functions of molecular chaperones. REFERENCES Ackermann, S. and A . Tzagoloff. 1990. ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F , - F Q complex. J. B i o l . C h e m . 265: 9952-9995. Atencio, D.P. and M.P. Yaffe. 1992. M A S 5 , a yeast homolog of DnaJ involved in mitochondrial protein import. M o l . C e l l . B i o l . 12: 283-291. Barraclough, R. and R.J. Ellis. 1980. Protein synthesis in chloroplasts. IX: Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. B i o c h e m . Biophys. A c t a 608: 19-31. Becker, K., B. Guiard, J. Rassow, T. Söllner, and K. Pfanner. 1992. Targeting of a chemically pure preprotein to mitochondria does not require the addition of a cytosolic signal recognition factor../. B i o l . C h e m . 267: 5637-5643. Blumberg, H. and P.A. Silver. 1991. A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast. N a t u r e 349: 627-630. Braig, K., J. Hainfeld, M . Simon, F. Furuya, and A . L . Horwich. 1993. Polypeptide bound to the chaperonin groEL binds within a central cavity. P r o c . N a t l . Acad. Sei. 90: 3978-3982. Buchwald, P., G . Krummeck, and G . Rödel. 1991. Immunological identification of yeast S C O l protein as a component of the inner mitochondrial membrane. M o l . G e n . Genei. 229:413-420. Bychkova, V . , R.H. Pain, and O.B. Ptitsyn. 1988. The "molten globule" State is involved in the translocation of proteins across membranes? F E B S L e u . 238: 231-234. Caplan, A . J . and M . G . Douglas. 1991. Characterization of YDJ1: A yeast homologue of the bacterial dnaJ protein. J. C e l l B i o l . 114: 609-621. Caplan, A . , D . M . Cyr, and M . G . Douglas. 1992a. Y D J l p facilitates Polypeptide translocation across different intracellular membranes by a conserved mechanism. C e l l 71: 1 143-1155. . 1993. Eukaryotic homologues of E s c h e r i c h i a coli DnaJ: A diverse protein fam- 78 T. Langer and W. Neupert ily that functions with HSP70 stress proteins. M o l . B i o l . C e l l 4: 555-563. Caplan, A . , J. Tsai, P J . Casey, and M.G. Douglas. 1992b. Farnesylation of Y D J l p is re- quired for function at elevated growth temperatures in Saccharomyces cerevisiae. J. B i o l . C h e m . 267: 18890-18895. Carbajal, E . , J.-F. Beaulieu, L . M . Nicole, and R.M. Tanguay. 1993. Intramitochondrial iocalization of the main 70-kDa heat-shock cognate protein in D r o s o p h i l a cells. E x p . C e l l Res. 207: 300-309. Chen, W.-J. and M . Douglas. 1987. The role of protein structure in the mitochondrial im- port pathway: Unfolding of mitochondrially bound precursors is required for membrane translocation./. B i o l . C h e m . 262: 15605-15609. . 1988. A n Fj-ATPase ß-subunit precursor lacking an internal tetramer-forming domain is imported into mitochondria in the absence of A T P . J. B i o l . C h e m . 263: 4997-5000. Cheng, M.Y., F.-U. Hartl, and A . L . Horwich. 1990. The mitochondrial chaperonin hsp60 is required for its own assembly. N a t u r e 348: 455-458. Cheng, M . Y . , F . - U . Hartl, J. Martin, R . A . Pollock, F. Kalousek, W. Neupert, E . M . Hallberg, R . L . Hallberg, and A . L . Horwich. 1989. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. N a t u r e 337: 620-625. Chirico, W . 1992. Dissociation of complexes between 70 kDa stress proteins and presecretory proteins is facilitated by a cytosolic factor. Biochem. Biophys. Res. C o m - m u n . 189: 1150-1156. Chirico, W., M . Waters, and G . Blobel. 1988. 70K heat-shock related proteins stimulated protein translocation into microsomes. N a t u r e 332: 805-810. Craig, E . A . , J. Kramer, J. Shilling, W . M . Werner, S. Holmes, S.J. Kosic, and C M . Nicolet. 1989. SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein. M o l . C e l l . B i o l . 9: 3000-3008. Cross, R. and C M . Nalin. 1981. Adenine nucleotide binding sites on beef heart F j ATPase. J. B i o l C h e m . 257: 2874-2881. Cyr, D., X . L u , and M . G . Douglas. 1992. Regulation of Hsp70 function by a eukaryotic DnaJ homolog. 7. B i o l C h e m . 267: 20927-20931. Cyr, D., R . A . Stuart, and W. Neupert. 1993. A matrix-ATP requirement for presequence translocation across the inner membrane of mitochondria. J. B i o l . Chem. 268: 23751-23754. Dingwall, C and R. Laskey. 1992. The nuclear membrane. Science 258: 942-947. Deshaies, R., B. Koch, M . Werner-Washburne, E. Craig, and R. Schekman. 1988. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor Polypeptides. N a t u r e 332: 800-805. Eilers, M . and G . Schatz. 1986. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. N a t u r e 322: 228-232. Gambill, B., W. Voos, P.J. Kang, B. Miao, T. Langer, E.A. A Craig, and K . Pfanner. 1993. dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J. C e l l B i o l 123: 9-26. Gasser, S., G . Daum, and G . Schatz. 1982. Import of proteins into mitochondria: Energydependent uptake of precursors by isolated mitochondria. J. Biol Chem. 257: 13034-13041. Georgopoulos, C , D. Ang, K . Liberek, and M . Zylicz. 1990. Properties of the Esc h e r i c h i a coli proteins heat shock proteins and their role in bacteriophage X growth. In i n biology and medicine Stress (ed. R.I. Morimoto et al.), pp. 191-222. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Chaperoning Mitochondrial Biogenesis 79 Gething, M . - J . and J. Sambrook, J. 1992. Protein folding in the cell. N a t u r e 355: 33-45. Glick, B. and G . Schatz. 1991. Import of proteins into mitochondria. A n n u . Rev. Geriet. 25: 21-44. Glick, B., A . Brandt, K. Cunningham, S. Müller, R. Hallberg, and G . Schatz. 1992. Cytochromes c and b are sorted to the intermembrane Space of yeast mitochondria by } 2 a stop-transfer mechanism. C e l l 69: 809-822. Goloubinoff, P., A . A . Gatenby, and G . H . Lorimer. 1989a. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. N a t u r e 337: 44-47. Goloubinoff, P., J.T. Christeller, A . A . Gatenby, and G . H . Lorimer. 1989b. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded State depends on two chaperonin proteins and M g A T P . N a t u r e 342: 884-889. Grivell, L . 1989. Nucleo-mitochondrial interactions in yeast mitochondrial biogenesis. 182: 477-493. E u r . J. Biochem. Hachiya, N . , R. Alam, Y . Sakasegawa, M . Sakaguchi, N . Mihara, and T . Omura. 1993. A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins. E M B O J. 12: 1579-1586. Hallberg, E . , Y . Shu, and R . L . Hallberg. 1993. Loss of mitochondrial hsp60 function: Nonequivalent effects on matrix-targeted and intermembrane-targeted proteins. M o l C e l l B i o l 13: 3050-3057. Hannavy K . , S. Rospert, and G . Schatz. 1993. Protein import into mitochondria: A paradigm for the translocation of Polypeptides across membranes. C u r r . B i o l 5: 694-700. Hartl, F . - U . and W. Neupert. 1990. Protein sorting to mitochondria: Evolutionary conservations of folding and assembly. Science 247: 930-938. Hartman, D., D. Dougan, N.J. Hoogenraad, and P.B. Hoj. 1992a. Heat shock proteins of barley mitochondria and chloroplasts. Identification of organellar hsplO and 12: Putative chaperonin 10 homologues. F E B S Lett. 305: 147-150. Hartman, D J . , N.J. Hoogenraad, R. Condron, and P.B. Hoj. 1992b. Identification of a mammalian 10-kDa heat shock protein, a mitochondrial chaperonin 10 homologue essential for assisted folding of trimeric Ornithine transcarbamoylase i n vitro. Acad. Proc. Natl Sei. 89: 3394-3398. Hemmingsen, S . M . , C . Woolford, S. van der Vies, K . Tilly, D . T . Dennis, C.P. Georgopoulos, R.W. Hendrix, and R.J. Ellis. 1988. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. N a t u r e 333: 330-334. Hendrick, J., and F . - U . Hartl. 1993. Molecular chaperone function of heat-shock proteins. A n n u . Rev. Biochem. 62: 349-384. Hendrix, R. 1979. Purification and properlies of GroE, a host protein involved in bacteriophage assembly. J. M o l . B i o l 129: 375-392. Hohn, T., B. Hohn, A . Engel, M . Wortz, and P.R. Smith. 1979. Isolation and characterization of the host protein GroE involved in bacteriophage X assembly. J. M o l B i o l 129: 359-373. Hutchinson, E . G . , W. Tichelaar, G . Hofhaus, H. Weiss, and K . R . Leonard. 1989. Identification and electron microsopic analysis of a chaperonin oligomer from crassa.EMBOJ. Neurospora 8: 1485-1490. Hwang, S. and G . Schatz. 1989. Translocation of proteins across the mitochondrial inner membrane, but not into the outer membrane, requires nucleotide triphosphates in the matrix. Proc. N a t l Acad. Sei 86: 8432-8436. Jindal, S., A . K . Dudani, B. Singh, C . B . Harley, and R.S. Gupta. 1989. Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen. M o l C e l l . B i o l 9: 2279-2283. 80 T. Langer and W. Neupert Johnson, R.B., K. Fearon, T. Mason, and S. Jindal. 1989. Cloning and characterization of the yeast chaperonin HSP60 gene. Gene 84: 295-302. Kang, P.-J., J. Ostermann, J. Shilling, W. Neupert, E. Craig, and N . Pfanner. 1990. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. N a t u r e 348: 137-142. Kiebler, M . , K. Becker, N. Pfanner, and W. Neupert. 1993. Mitochondrial protein import: Specific recognition and membrane translocation of preproteins. J. M e m b r . B i o l . 135: 191-207. Kim, P. and R.L. Baldwin. 1990. Intermediates in the folding reactions of small proteins. 59: 631-660. A n n u . Rev. Biochem. Koll, H . , B. Guiard, J. Rassow, J. Ostermann, A . Horwich, W. Neupert, and F . - U . Hartl. 1992. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane Space. C e l l 68: 1163-1175. Kurihara, T. and P.A. Silver. 1992. DnaJ homologs and protein transport. In M e m b r a n e biogenesis and protein targeting (ed. W. Neupert and R. Lill), pp. 309-384. Elsevier, New York. Langer, T., G . Pfeifer, J. Martin, W. Baumeister, and F . - U . Hartl. 1992a. Chaperoninmediated protein folding: GroES binds to one end of the G r o E L cylinder, which accommodates the protein Substrate within its central cavity. E M B O J. 11: 4757-5765. Langer, T., C. L u , H . Echols, J. Flanagan, M . Hayer-Hartl, and F.-U.Hartl. 1992b. Successive action of DnaK, DnaJ and G r o E L along the pathway of chaperone-mediated protein folding. N a t u r e 356: 683-689. Liberek, K., J. Marszalek, D. Ang, C. Georgopoulos, and M . Zylicz. 1991. coli Proc. Escherichia DnaJ and GrpE heat shock proteins joinlly stimulate ATPase activity of DnaK. N a t l . Acad. Sei. 88: 2874-2878. Lill, R., R. Stuart, M . Drygas, F. Nargang, and W. Neupert. 1992a. Import of cytochrome c heme lyase into mitochondria: A novel pathway into the intermembrane Space. EMBOJ. 11: 449-456. Lill, R., C . Hergersberg, H . Schneider, T. Söllner, R. Stuart, and W. Neupert. 1992b. General and exceptional pathways of protein import into the sub-mitochondrial compartments. In M e m b r a n e biogenesis and protein targeting (ed. W. Neupert and R. Lill), pp. 265-278. Elsevier, New York. Lubben, T . H . , A . A . Gatenby, G . K . Donaldson, G . H . Lorimer, and P.V. Viitanen. 1990. Identification of a groES-like chaperonin in mitochondria that facilitates protein folding. Proc. N a t l . Acad. Sei. 87: 7683-7687. Luis, A . M . , A . Alconada, and J . M . Cuezva. 1990. The alpha regulatory subunit of the mitochondrial F,-ATPase complex is a heat-shock protein. Identification of two highly conserved amino acid sequences among the alpha-subunits and molecular chaperones. J. B i o l . C h e m . 265: 7713-7716. Manning-Krieg, U . , P. Scherer, and G . Schatz. 1991. Sequential action of mitochondrial chaperones in protein import into the matrix. E M B O J. 10: 3273-3280. Martel, R., L.P. Cloney, L . E . Pelcher, and S . M . Hemmingsen. 1990. Unique composition of plastid chaperonin-60: a and ß polypeptide-encoding genes are highly divergent. G^e 94: 181-187. Martin, J., A . L . Horwich, and F . - U . Hartl. 1992. Prevention of protein denaturation under heat stress by the chaperonin Hsp60. Science 258: 995-998. Martin, J., K. Mahlke, and N . Pfanner. 1991a. Role of an energized inner membrane in mitochondrial protein import./. B i o l . C h e m . 266: 18051-18057. Martin, J., T. Langer, R. Boteva, A . Schramel, A . Horwich, and F . - U . Hartl. 1991b. Chaperonin-mediated protein folding at the surface of groEL through a "molten Chaperoning Mitochondrial Biogenesis 81 globule"-like intermediate. N a t u r e 352: 36-42. Mayer, A . , R. Lill, and W. Neupert. 1993. Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria. J. C e l l B i o l 121: 2233-2243. McMullin, T . W . and R.L. Hallberg. 1987. A normal mitochondrial protein is selectively synthesized and accumulated during heat shock in T e t r a h y m e n a thermophila. Mol C e l l B i o l 7: 4414-4423. . 1988. A highly evolutionary conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene. M o l Cell Biol 8: 371-380. Mizzen, L.A., C. Chang, J.I. Garrels, and W.J. Welch. 1989. Identification, characteriza- tion, and purification of two mammalian stress proteins present in mitochondria, grp75, a member of the hsp70 family and hsp58, a homolog of the bacterial G r o E L protein. J. B i o l C h e m . 264: 20664-20675. Murakami, H . , D. Pain, and G . Blobel. 1988. 70K heat-shock related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria./. C e l l B i o l . 107: 2051-2057. Murakami, K. and M . Mori. 1990. Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein. E M B O J. 9: 3201-3208. Murakami, K . , S. Tanase, Y . Morino, and. N . Mori. 1992. Presequence binding factordependent and -independent import of proteins into mitochondria../. B i o l . C h e m . 267: 13119-13122. Neupert, W . and N . Pfanner. 1993. Roles of molecular chaperones in protein targeting to mitochondria. Philos. Neupert, W., F.-U. T r a n s . R . Soc. Lond. 339: 355-362. Hartl, E . A . Craig, and N. Pfanner. 1990. How do Polypeptides cross the mitochondrial membranes? C e l l 63: 447-450. Nicholson, D., C . Hergersberg, and W. Neupert. 1988. Role of cytochrome c heme lyase in the import of cytochrome c into mitochondria../. B i o l C h e m . 263: 19034-19042. Ono, H . and S. Tuboi. 1988. The cytosolic factor required for import of precursors of mitochondrial precursor proteins into mitochondria./. B i o l C h e m . 263: 3188-3193. . 1990. Purification and identification of a cytosolic factor required for import of precursors of mitochondria proteins into mitochondria. A r c h . Biochem. Biophys. 280: 299-304. Ostermann, J., A . Horwich, W. Neupert, and F . - U . Hartl. 1989. Protein folding in mitochondria requires complex formation with hsp60 and A T P hydrolysis. N a t u r e 341: 125-130. Ostermann, J., W. Voss, P. Kang, E . Craig, W. Neupert, and N. Pfanner. 1990. Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix. F E B S L e t t . 2 1 1 : 281-284. Palleros, D.R., W.J. Welch, and A . L . Fink. 1991. Interaction of hsp70 with unfolded proteins: Effects of temperature and nucleotides on the kinetics of binding. Proc. Acad. Natl Sei. 88: 5719-5723. Pfanner, N . , M . Tropschug, and W. Neupert. 1987. Mitochondrial protein import: Nucleoside triphosphates are involved in conferring import-competence to precursors. C e l l 4 9 : 815-823. Pfanner, N . , J. Rassow, B. Guiard, T. Söllner, F.-U. Hartl, and W . Neupert. 1990. Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria./. B i o l . C h e m . 265: 16324-16329. Picketts, D J . , C.S.K. Mayanil, and R.S. Gupta. 1989. Molecular cloning of a Chinese hamster mitochondrial protein related to the "chaperonin" family of bacterial and plant 82 T. Langer and W. Neupert proteins. J. B i o l . C h e m . 264: 12001-12008. Prasad, T.K., E . Hack, and R . L . Hallberg. 1990. Function of the maize mitochondrial chaperonin hsp60: Specific association between hsp60 and newly synthesized F j ATPase alpha subunits. M o l C e l l . B i o l . 10: 3979-3986. Rassow, J., F . - U . Hartl, B. Guiard, N . Pfanner, and W. Neupert. 1990. traverse the mitochondrial envelope in an extended State. F E B S Lett. Polypeptides 275: 190-194. Rassow, J., B. Guiard, U . Wienhues, V . Herzog, F . - U . Hartl, and W. Neupert. 1989. Translocation arrest by reversible folding of a precursor protein imported into mitochondria: A means to quantitate translocation contact sites. J. C e l l B i o l . 109: 1421-1428. Reading, D.S., R . L . Hallberg, and A . M . Meyers. 1989. Characterization of the yeast H S P 6 0 gene coding for a mitochondrial assembly factor. N a t u r e 337: 655-659. Scherer, P.E., U . C . Krieg, S.T. Hwang, D. Vestweber, and G . Schatz. 1990. A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. E M B O J. 9: 4315-4322. Schleyer, M . and W. Neupert. 1982. Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. E u r . J. Biochem. 125: 109-116. . 1985. Transport of proteins into mitochondria: Translocation intermediates spanning contact sites between inner and outer membranes. C e l l 43: 330-350. Schwerzmann, K . , L . M . Cruz-Orive, R. Eggmen, A . Sänger, and E.R. Weibel. 1986. Molecular architecture of the inner membrane of mitochondria from rat liver: A combined biochemical and stereological study. J. C e l l B i o l 102: 97-103. Segui-Real, B., R. Stuart, and W. Neupert. 1992. Transport of proteins into the various subcompartments of mitochondria. F E B S Lett. 313: 2-7. Sheffield, W . , G . Shore, and S. Randall. 1990. Mitochondrial protein import: Effects of 70-kd hsp70 on Polypeptide folding, aggregation and import competence. J. B i o l C h e m . 265: 11069-11076. Skerjanc, L S . , W.P. Sheffield, S.K. Randall, J.R. Silvius, and G . Shore. 1990. Import of precursor proteins into mitochondria: Site of Polypeptide unfolding. J. B i o l C h e m . 265, 9444-9451. Smith, B. and M.P. Yaffe. 1991. A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle. M o l C e l l B i o l . 11: 2647-2655. Stuart, R. and W. Neupert. 1990. Apocyptochrome c: An exceptional mitochondrial precursor protein using an exceptional import pathway. B i o c h i m i e 72: 115-121. Stuart, R.A., A . Gruhler, I. van der Klei, B. Guiard, H . Koll, and W. Neupert. 1994. The requirement of matrix A T P for the import of precursor proteins into the mitochondrial matrix and intermembrane Space. E u r . J. Biochem. Touchette, N . A . , K . M . tase from Escherichia (in press). Perry, and C R . Matthews. 1986. Folding of dihydrofolate reduccoli. Biochemistry 25: 5445-5452. Vestweber, D. and G . Schatz. 1988. Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria. E M B O J . 7: 1147-1151. Voos, W . , B . D . Gambill, B. Guiard, K . Pfanner, and E . A . Craig. 1993. Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. J. C e l l B i o l . 123: 109-118. Waegemann, K . , H . Paulsen, and J. Soll. 1990. Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence. F E B S Lett. 261: 89-92. Wickner, S., J. Hoskins, and K . McKenney. 1991. Function of DnaJ and DnaK as chaperones in origin-specific D N A binding by RepA. N a t u r e 350: 165-167. Chaperoning Mitochondrial Biogenesis 83 VWienhues, U . , K. Becker, M . Schleyer, B. Guiard, M . Tropschug, A . Horwich, N . Pfanner, and W. Neupert. 1991. Protein folding causes an arrest of preprotein translocation in mitochondria in vivo. J. C e l l B i o l 115: 1601-1609. ZZhong, T. and K . T . Arndt. 1993. The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation. C e l l 73: 1175-1186. Ziylicz, M . , D. Ang, K. Liberek, and C. Georgopoulos. 1989. Initiation of lambda D N A replication with purified host- and bacteriophage-encoded proteins: The role of the dnaK, dnaJ and grpE heat shock proteins. E M B O J . 8: 1601-1608.
© Copyright 2016 ExploreDoc