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Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1.

ABSTRACT: Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge-charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1-Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an "anchoring and docking" model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.

SUBMITTER: Qian X

PROVIDER: S-EPMC1222294 | biostudies-other | 2002 Jan

REPOSITORIES: biostudies-other

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Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p.

Project description:Organisms encode multiple homologous heat shock protein (Hsp)-70s, which are essential protein chaperones that play the major role in cellular protein "quality control." Although Hsp70s are functionally redundant and highly homologous, many possess distinct functions. A regulatory motif underlying such distinctions, however, is unknown. The 98% identical cytoplasmic Hsp70s Ssa1p and Ssa2p function differently with regard to propagation of yeast [URE3] prions and in the vacuolar-mediated degradation of gluconeogenesis enzymes, such as FBPase. Here, we show that the Hsp70 nucleotide binding domain (NBD) regulates these functional specificities. We find little difference in ATPase, protein refolding, and amyloid inhibiting activities of purified Ssa1p and Ssa2p, but show that interchanging NBD residue alanine 83 (Ssa1p) and glycine 83 (Ssa2p) switched functions of Ssa1p and Ssa2p in [URE3] propagation and FBPase degradation. Disrupting the degradation pathway did not affect prion propagation, however, indicating these are two distinct processes where Ssa1/2p chaperones function differently. Our results suggest that the primary evolutionary pressure for Hsp70 functional distinctions is not to specify interactions of Hsp70 with substrate, but to specify the regulation of this activity. Our data suggest a rationale for maintaining multiple Hsp70s and suggest that subtle differences among Hsp70s evolved to provide functional specificity without affecting overall enzymatic activity.

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Synergistic binding of DnaJ and DnaK chaperones to heat shock transcription factor σ32 ensures its characteristic high metabolic instability: implications for heat shock protein 70 (Hsp70)-Hsp40 mode of function.

Project description:Escherichia coli heat shock transcription factor σ(32) is rapidly degraded by ATP-dependent proteases, such as FtsH and ClpYQ. Although the DnaK chaperone system (DnaK, DnaJ, and GrpE) promotes σ(32) degradation in vivo, the precise mechanism that is involved remains unknown. Our previous results indicated that σ(32) mutants containing amino acid substitution in the N-terminal half of Region 2.1 are markedly stabilized in vivo. Here, we report the further characterization of these mutants by examining purified σ(32) mutants in vitro. Surprisingly, I54A σ(32), a very stable mutant, is more susceptible to ClpYQ and FtsH proteases than wild-type σ(32), indicating that the stability of σ(32) does not always reflect its susceptibility to proteases. Co-precipitation and gel filtration analyses show that purified σ(32) mutants exhibit a reduced affinity for DnaJ, leading to a marked decrease in forming a complex with DnaK in the presence of DnaJ and ATP. Other mutants with modestly increased stability (A50S σ(32) and K51E σ(32)) show an intermediate efficiency of complex formation with DnaK, suggesting that defects in binding to DnaK and DnaJ are well correlated with metabolic stability; effective interaction with DnaK promotes σ(32) degradation in vivo. We argue that the stable and effective interaction of heat shock protein 70 (Hsp70) with a substrate polypeptide may generally require the simultaneous binding of heat shock protein 40 (Hsp40) to distinct sites on the substrate.

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Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

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Project description: Not available

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