The Yeast Hsp110 Sse1 Functionally Interacts with the Hsp70 Chaperones Ssa and Ssb

Abstract

There is growing evidence that members of the extended Hsp70 family of molecular chaperones, including the Hsp110 and Grp170 subgroups, collaborate in vivo to carry out essential cellular processes. However, relatively little is known regarding the interactions and cellular functions of Sse1, the yeast Hsp110 homolog. Through co-immunoprecipitation analysis, we found that Sse1 forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. Binding of Ssa or Ssb to Sse1 was mutually exclusive. The ATPase domain of Sse1 was found to be critical for interaction as inactivating point mutations severely reduced interaction with Ssa and Ssb. Sse1 stimulated Ssa1 ATPase activity synergistically with the co-chaperone Ydj1, and stimulation required complex formation. Ssa1 is required for post-translational translocation of the yeast mating pheromone ␣-factor into the endoplasmic reticulum. Like ssa mutants, we demonstrate that sse1⌬ cells accumulate prepro-␣-factor, but not the co-translationally imported protein Kar2, indicating that interaction between Sse1 and Ssa is functionally significant in vivo. These data suggest that the Hsp110 chaperone operates in concert with Hsp70 in yeast and that this collaboration is required for cellular Hsp70 functions.Cells respond to protein-denaturing stresses such as heat by rapidly inducing expression of a wide array of heat shock genes. Chief among these are the molecular chaperones, highly conserved proteins that associate with and protect unfolded proteins, preventing their aggregation and supporting refolding (1). Perhaps the most abundant and well characterized chaperones are the heat shock protein 70 (Hsp70) 2 family, found in all cell types from bacteria to eukaryotes (2, 3). Hsp70s assist in protein refolding by binding of exposed hydrophobic surfaces of a substrate to a C-terminal peptide binding domain. Cycles of substrate binding and release are brought about by transition between low and high affinity binding states regulated by nucleotide occupancy in the N-terminal ATPase domain (4).In eukaryotic cells Hsp70 class chaperones can be divided into three subfamilies: 1) DnaK-like, 2) Hsp110, and 3) Grp170 (5). The budding yeast, Saccharomyces cerevisiae, possesses 14 Hsp70 homologs with family members present in the cytoplasm, endoplasmic reticulum (ER), and mitochondria (6). The most well studied Hsp70s in yeast are the cytoplasmic Ssa proteins (stress seventy A), encoded by the differentially expressed SSA1-4 genes. Ssa1-4 (collectively referred to as "Ssa") perform largely redundant functions and the presence of at least one SSA gene is required for viability (7). Ssa chaperones are involved in cellular processes such as translation, translocation of proteins across cellular membranes, and general protein folding (8). Cells depleted of Ssa exhibit multiple cellular defects, including: (i) growth arrest in G 2 /M phase, (ii) accumulation of precursor pr...

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

“…6B). As we previously demonstrated, Sse1 strongly interacted with both Hsp70s (22). In contrast, Fes1 appeared to interact exclusively with Ssa in vivo, with only background amounts of Ssb co-purifying.…”

“…Sse1 has been implicated in Hsp70-mediated protein folding at the ribosome, Hsp90 chaperoning of signal transduction, and post-translational translocation of pre-pro ␣-factor (21,22,37,38). Both Sse1 and Fes1 participate in Hsp70-dependent ubiquitination and degradation of misfolded proteins (39 -43).…”

Unique Peptide Substrate Binding Properties of 110-kDa Heat-shock Protein (Hsp110) Determine Its Distinct Chaperone Activity

“…A number of studies suggested that Hsp110s participate in many processes associated with cytosolic Hsp70s, including de novo protein folding, refolding under stress, translocation into the endoplasmic reticulum, degradation, and prion formation (24 -31). Consistent with these observations, Hsp110s have recently been shown to form complexes with cytosolic Hsp70s for which they function as the major NEF (25,(32)(33)(34)(35). Moreover, the corresponding mechanism of this nucleotide exchange activity was recently revealed by a number of biochemical and crystallographic studies (36 -39).…”

Hsp110 Cooperates with Different Cytosolic HSP70 Systems in a Pathway for de Novo Folding

“…Interestingly, mammalian Hsp110 has been reported to inhibit the ATPase activity of nucleotide-loaded Hsp70 (29), which could be consistent with an Hsp110 nucleotide exchange activity. In addition, Shaner et al (49) report that Sse1p can stimulate the ATPase of SSA in conjunction with its J-protein Ydj1. Because nucleotide exchange by Hsp70 promotes polypeptide release, the suggestion that Sse1p may act as a nucleotide exchange factor is consistent with our functional analysis.…”