The Mitochondrial hsp70 Chaperone System

Abstract

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.

Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling

“…We found that ⌬F(n) is, at most, 3 kcal͞mol when n is small (Fig. 3A), whereas the affinity of Hsp70 in the ADP form (locked state) for its substrates has been experimentally measured to be ⌬H Ϸ Ϫ9 kcal͞mol (20,26,34,35). Therefore, the constraint is verified, and the locking of Hsp70 onto its substrate is a thermodynamically favorable process.…”

Substrate Binding Induces Depolymerization of the C-terminal Peptide Binding Domain of Murine GRP78/BiP

“…with those obtained with three other HSP70 proteins: cognate hsc70 (51-53), E. coli DnaK (25,54), and mitochondrial hsp70 (mhsp70) (55), which demonstrate the coexistence of multiple oligomeric species in HSP70 preparations, all in equilibrium with each other, in a process dependent on temperature, concentration, adenine nucleotides, peptidic substrates, and accessory proteins. Self-association thus appears to be a general property shared by all members of the HSP70 family and should be taken into account in biochemical studies of the HSP70 functional cycle, as the different oligomeric species may have different affinities and on/off rates for nucleotidic and peptidic substrates as reported for hsc70 (52).…”

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

“…Thus, it seems inappropriate at this time to form rigid models of the priming step based on stoichiometry data that require the action of specifically a monomer or a dimer of hsp70 on the receptor. It is possible that only one molecule of hsp70 is bound directly to and interacting productively with the receptor, and that under these conditions of assembly with purified proteins the receptor-bound hsp70 can associate with other molecules of hsp70 to form dimers and trimers, much as purified hsp70 and its homologs have been shown to do in solution (32)(33)(34). It is perhaps important to note that analysis of both the native and assembled hsp90⅐Hop⅐hsp70 machinery has revealed a molar ratio of 1 hsp70 per hsp90 dimer (6,18).…”

“…Bacterial DnaK and its mammalian mitochondrial and cytoplasmic homologs self-associate in solution into dimers, trimers, and probably higher oligomers, with the equilibrium between these forms being dependent upon the concentration of the chaperone and the equilibrium being shifted toward the monomer by ATP or binding of peptide substrate (32)(33)(34). Under concentrated conditions, our purified hsp70 is predominantly dimeric on native gel electrophoresis with some trimers being detectable, yet under dilute conditions in reticulocyte lysate, the free hsp70 behaves as a monomer on molecular sieve chromatography (6), as did our purified hsp70 (data not shown).…”