The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1⌬ yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1⌬ and lethality of sse1⌬sse2⌬ cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1⌬ growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

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...

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high affinity (K d~1 0 −8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones, and that ATP binding to Sse1 is required for binding to Hsp70s. Sse1•Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity, and are sufficient to mediate heterodimerization with no discernable contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.Molecular chaperones of the Hsp70 class are present in all cells and are essential for tolerance of protein denaturing stresses as well as protein biogenesis and regulation under normal growth conditions. Hsp70s share a common architecture defined by an amino-terminal nucleotide binding/ATPase domain (NBD) that regulates chaperone activity of a carboxyl-terminal peptide peptide binding domain (PBD). Hsp70 chaperones bind to exposed hydrophobic segments of proteins through the PBD, preventing their aggregation and assisting in folding to achieve native conformation. Hsp70 ATPase activity governs reversible transition between low-and high-affinity substrate binding states, and is in turn regulated by additional factors (reviewed in (1)). Hsp40-type, or J-domain co-chaperones stimulate ATP hydrolysis, and cochaperones such as GrpE in bacteria and Fes1 in yeast accelerate release of nucleotide from Hsp70 (2-4). There is increasing evidence for collaboration of Hsp70s with co-chaperones that are themselves divergent Hsp70 homologs. For example, in the yeast endoplasmic reticulum (ER), the lumenal Hsp70, Kar2, binds the Grp170 subfamily homolog Lhs1, leading to reciprocal regulation of their respective ATPase activities (5). Similarly, Ssb ATPase activity is stimulated by a heterodimeric complex known as RAC (ribosome-associated complex) consisting of the Hsp70 Ssz1 and the Hsp40 Zuo1 (6,7). The Hsp110 subfamily of Hsp70-related chaperones is a poorly understood group defined by an extended linker region separating the ß and α subdomains of the PBD, as well as an extended carboxy terminus of unknown significance (8). Murine Hsp105α was shown to inhibit Hsp70-mediated refolding of a model substrate by inhibiting Hsp70 ATPase activity in vitro (9,10). However, the molecular mech...

Aerobic organisms possess efficient systems for the transport of copper. This involves transporters that mediate the passage of copper across biological membranes to reach essential intracellular copper-requiring enzymes. In this report, we identify a new copper transporter in Schizosaccharomyces pombe, encoded by the ctr6 ؉ gene. The transcription of ctr6 ؉ is induced under copper-limiting conditions. This regulation is mediated by the cis-acting promoter element CuSE (copper-signaling element) through the copper-sensing transcription factor Cuf1. An S. pombe strain bearing a disrupted ctr6⌬ allele displays a strong reduction of copper,zinc superoxide dismutase activity. When the ctr6؉ gene is overexpressed from the thiamine-inducible nmt1 ؉ promoter, the cells are unable to grow on medium containing exogenous copper. Surprisingly, this copper-sensitive growth phenotype is not due to an increase of copper uptake at the cell surface. Instead, copper delivery across the plasma membrane is reduced. Consistently, this results in repressing ctr4 ؉ gene expression. By using a functional ctr6 ؉ epitope-tagged allele expressed under the control of its own promoter, we localize the Ctr6 protein on the membrane of vacuoles. Furthermore, we demonstrate that Ctr6 is an integral membrane protein that can trimerize. Moreover, we show that Ctr6 harbors a putative copper-binding Met-X-His-Cys-X-Met-X-Met motif in the amino terminus, which is essential for its function. Our findings suggest that under conditions in which copper is scarce, Ctr6 is required as a means to mobilize stored copper from the vacuole to the cytosol.

Divergent relatives of the Hsp70 protein chaperone such as the Hsp110 and Grp170 families have been recognized for some time, yet their biochemical roles remained elusive. Recent work has revealed that these "atypical" Hsp70s exist in stable complexes with classic Hsp70s where they exert a powerful nucleotide-exchange activity that synergizes with Hsp40/DnaJ-type cochaperones to dramatically accelerate Hsp70 nucleotide cycling. This represents a novel evolutionary transition from an independent protein-folding chaperone to what appears to be a dedicated cochaperone. Contributions of the atypical Hsp70s to established cellular roles for Hsp70 now must be deciphered.