During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent "translocation motor" that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.

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The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, binds hydrophobic polypeptide segments in extended conformation. The co-chaperone DnaJ (Hsp40) has been reported to bind native and denatured proteins as well as peptides. We tested pseudopeptides of D-amino Chaperones of the Hsp70 1 family fulfill essential functions in protein folding by preventing and reversing off-pathway interactions that lead to aggregation (1, 2). Hsp70s are also required for membrane translocation of precursor polypeptides (3, 4) and participate in the degradation of misfolded proteins (2).The Hsp70 chaperone system of Escherichia coli comprises the Hsp70 homolog DnaK and the co-chaperones DnaJ and GrpE. DnaK consists of a 44-kDa ATPase domain, the crystal structure of which has recently been determined in a complex with a dimer of truncated GrpE (5), and a 27-kDa peptidebinding domain, the crystal structure of which has been solved in a complex with a synthetic peptide ligand (6). The chaperone effects of the DnaK/DnaJ/GrpE system are based on the ability of DnaK to bind extended hydrophobic segments of proteins in a reversible manner and possibly to exert conformational work on them (2, 7). Binding and release of polypeptides are modulated by ATP binding and hydrolysis (8), which on their part are controlled by the co-chaperones GrpE and DnaJ, respectively (7, 9). DnaJ interacts with DnaK through its highly conserved N-terminal J-domain (10, 11). However, DnaJ appears to exert also a chaperone function of its own (12). The co-chaperone associates with denatured polypeptides independently of DnaK, e.g. luciferase and rhodanese (13, 14), preventing their aggregation and targeting them to DnaK.In previous studies, we have found various peptides derived from the presequence of mitochondrial aspartate aminotransferase to be high affinity ligands of DnaK (7).2 In a search for peptide mimetics that might be used as inhibitors of the chaperone system, we tested normal all-D and retro all-D (retroinverso) analogs of a parent all-L peptide for binding to DnaK and DnaJ. In the normal all-D peptide mimetics, all L-amino acid residues have been replaced by the corresponding D-enantiomers; the main chains of these peptide analogs have the same direction as in the parent peptides, whereas the side chains occupy enantiomorphic positions. In contrast, retro all-D peptides, i.e. mimetic peptides of D-amino acids with, as compared with the reference L-peptides, reverse sequence, exhibit in extended conformation the same side chain topology as their native counterparts (15). In certain cases they may therefore retain the biological activity of the reference L-peptides while being much less susceptible to proteolytic degradation (16 -18).Here we show that the co-chaperone DnaJ binds the tested pseudo-peptides with similar affinities as the parent all-L peptide. In contrast, DnaK did not bind any of the D-peptides with measurable affinity. EXPERIMENTAL PROCEDURESPurification of Proteins-DnaK from an overproducing strain of E. coli bearin...

Mitochondrial hsp70 (mhsp70) is a key component in the import and folding of mitochondrial proteins. In both processes, mhsp70 cooperates with the mitochondrial nucleotide exchange factor mGrpE (also termed Mge1p). In this work we have characterized the selfassociation of purified mhsp70, the interaction of mhsp70 with isolated mGrpE and protein substrate, and the effect of nucleotides on these interactions. mhsp70 can form oligomers that are dissociated by ATP or by a nonhydrolyzable ATP analog. A substrate peptide binds to mhsp70 in the absence of added nucleotides and is released by ATP but not by ADP. Binding of the peptide causes nucleotide-independent dissociation of the mhsp70 oligomers and enhances the mhsp70 ATPase. Purified mGrpE forms a homodimer. In the absence of added nucleotides, one mGrpE dimer binds to one molecule of mhsp70, forming a stable 122 kDa hetero-oligomer. This complex is weakened by ADP and completely dissociated by ATP.Members of the 70-kDa heat shock protein (hsp70) family play an essential role in protein folding, transport of proteins into different cellular compartments, and regulation of the heat shock response (1, 2). The members of this protein family are highly conserved from bacteria to man (3, 4). In eukaryotes, at least one hsp70 is uniquely located in the mitochondrial matrix. In the yeast Saccharomyces cerevisiae the product of the SSC1 gene, mitochondrial hsp70 (mhsp70), 1 is essential for growth and plays a major role in the import and folding of mitochondrial proteins (5-9). Recently it was shown that mhsp70 forms a transient complex with its membrane anchor Tim44 and mitochondrial GrpE (mGrpE; Refs. 10 -12). As the translocating polypeptide chain emerges in the matrix, mhsp70 binds to it in an ATP-dependent manner, and ATP hydrolysis by mhsp70 then drives the inward movement of the polypeptide chain (13,14).Although the different functions of mhsp70 have been extensively studied, very little is known about the properties of the purified protein, its interaction with nucleotides and protein substrates, and the effect of co-chaperones on these interactions. Information on these points will be essential for understanding how mhsp70 mediates the translocation of polypeptides across the mitochondrial inner membrane as well as the folding of some of these polypeptides in the mitochondrial matrix. To address these questions, we purified mhsp70 from yeast and studied its oligomeric state; we also explored the effect of nucleotides and a peptide substrate on mhsp70 oligomerization. Finally, we determined the N terminus of mature mGrpE, expressed the mature protein in Escherichia coli, and assayed its interaction with mhsp70.

DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the ␥-phosphate cleavage of DnaKbound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured luciferase, thus form ternary complexes with DnaJ and DnaK. A previous study has shown that D-peptides compete with L-peptides for the same binding site in DnaJ but do not bind to DnaK (Feifel, B., Schö nfeld, H.-J., and Christen, P. (1998) J. Biol. Chem. 273, 11999 -12002). Here we report that D-peptides efficiently inhibit the refolding of denatured luciferase by the DnaK/DnaJ/GrpE chaperone system (EC 50 ‫؍‬ 1-2 M). The inhibition of the chaperone action is due to the binding of D-peptide to DnaJ (K d ‫؍‬ 1-2 M), which seems to preclude DnaJ from forming ternary (ATP⅐DnaK) m ⅐substrate⅐DnaJ n complexes. Apparently, simultaneous binding of DnaJ and DnaK to one and the same target polypeptide is essential for effective chaperone action. The Hsp701 chaperone system of Escherichia coli includes DnaK and the two cohort proteins: DnaJ, a Hsp40 homolog, and GrpE. The chaperones assist protein folding by preventing and reversing off-pathway interactions that lead to aggregation (1). The key features of the Hsp70 chaperone system are the binding of unfolded hydrophobic segments of the target polypeptides to the ATP-liganded form of DnaK, the stabilization of the complex upon ATP hydrolysis, and the release of the bound ligands upon ADP/ATP exchange (1-3). This binding/ release cycle is controlled by DnaJ and the nucleotide exchange factor GrpE (4, 5). DnaJ interacts with DnaK through its highly conserved NH 2 -terminal J-domain and stimulates the hydrolysis of DnaK-bound ATP (2, 6). DnaJ also exerts a chaperone action on its own; upon association with denatured polypeptides, such as luciferase or rhodanese, it may prevent their aggregation (3, 6). Recently, it has been shown that D-peptides bind to DnaJ but not to DnaK (7,8). D-Peptides bind to the same site of DnaJ as L-peptides (7). Here we report that two retro-all D-peptides derived from the NH 2 -terminal segment of rhodanese inhibit the DnaK/DnaJ/GrpE chaperone system in refolding denatured firefly luciferase. EXPERIMENTAL PROCEDURESProteins-DnaK was isolated from an overproducing strain of E. coli (JM 83) bearing the plasmid pTPG9 (3). The stock solution of the protein in assay buffer (25 mM Hepes/NaOH, 100 mM KCl, pH 7.0) was stored at Ϫ80°C and contained less than 0.1 mol of ADP/mol of DnaK (9). The concentration of DnaK was determined photometrically with ⑀ 280 ϭ 14.6 mM Ϫ1 cm Ϫ1 . DnaJ and GrpE were prepared as described (10); stock solutions were stored at Ϫ80°C in 50 mM Tris/HCl, 100 mM NaCl at pH 7.7.Peptides-The peptide ala-p5 (ALLLSAPRR) was purchased with a purity of Ͼ90% from Chiron. The peptide was dissolved in 0.1% (v/v) acetic acid, 10% (v/v) acetonitrile and stored at Ϫ20°C. The two Dpeptides RI1-17 (...