Molecular chaperones are a functionally defined set of proteins which assist the structure formation of proteins in vivo. Without certain protective mechanisms, such as binding nascent polypeptide chains by molecular chaperones, cellular protein concentrations would lead to misfolding and aggregation. In the mammalian system, the molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins, like steroid hormone receptors, transcription factors, and kinases, some of which are involved in cancer progression. Hsp70 and Hsp90 form a multichaperone complex, in which both are connected by a third protein called Hop. The connection of and the interplay between the two chaperone machineries is of crucial importance for cell viability. This review provides a detailed view of the Hsp70 and Hsp90 machineries, their cofactors and their mode of regulation. It summarizes the current knowledge in the field, including the ATP-dependent regulation of the Hsp70/Hsp90 multichaperone cycle and elucidates the complex interplay and their synergistic interaction.

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...

The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer. p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner. The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo. Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail. Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity. Specifically, the DNAbinding domain of p53 and the middle and carboxyterminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation. Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation.

Ppt1 is the yeast member of a novel family of protein phosphatases, which is characterized by the presence of a tetratricopeptide repeat (TPR) domain. Ppt1 is known to bind to Hsp90, a molecular chaperone that performs essential functions in the folding and activation of a large number of client proteins. The function of Ppt1 in the Hsp90 chaperone cycle remained unknown. Here, we analyzed the function of Ppt1 in vivo and in vitro. We show that purified Ppt1 specifically dephosphorylates Hsp90. This activity requires Hsp90 to be directly attached to Ppt1 via its TPR domain. Deletion of the ppt1 gene leads to hyperphosphorylation of Hsp90 in vivo and an apparent decrease in the efficiency of the Hsp90 chaperone system. Interestingly, several Hsp90 client proteins were affected in a distinct manner. Our findings indicate that the Hsp90 multichaperone cycle is more complex than was previously thought. Besides its regulation via the Hsp90 ATPase activity and the sequential binding and release of cochaperones, with Ppt1, a specific phosphatase exists, which positively modulates the maturation of Hsp90 client proteins.

The molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop. In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction. With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins. Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro. The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90. Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system. Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1. Thus, Sti1 is a potent novel effector for the Hsp70 ATPase.