Diverse Cellular Functions of the Hsp90 Molecular Chaperone Uncovered Using Systems Approaches

SummaryA comprehensive understanding of the cellular functions of the Hsp90 molecular chaperone has remained elusive. Although Hsp90 is essential, highly abundant under normal conditions, and further induced by environmental stress, only a limited number of Hsp90 “clients” have been identified. To define Hsp90 function, a panel of genome-wide chemical-genetic screens in Saccharomyces cerevisiae were combined with bioinformatic analyses. This approach identified several unanticipated functions of Hsp90 under normal conditions and in response to stress. Under normal growth conditions, Hsp90 plays a major role in various aspects of the secretory pathway and cellular transport; during environmental stress, Hsp90 is required for the cell cycle, meiosis, and cytokinesis. Importantly, biochemical and cell biological analyses validated several of these Hsp90-dependent functions, highlighting the potential of our integrated global approach to uncover chaperone functions in the cell

Heterozygous Yeast Deletion Collection Screens Reveal Essential Targets of Hsp90

Hsp90 is an essential eukaryotic chaperone with a role in folding specific “client” proteins such as kinases and hormone receptors. Previously performed homozygous diploid yeast deletion collection screens uncovered broad requirements for Hsp90 in cellular transport and cell cycle progression. These screens also revealed that the requisite cellular functions of Hsp90 change with growth temperature. We present here for the first time the results of heterozygous deletion collection screens conducted at the hypothermic stress temperature of 15°C. Extensive bioinformatic analyses were performed on the resulting data in combination with data from homozygous and heterozygous screens previously conducted at normal (30°C) and hyperthermic stress (37°C) growth temperatures. Our resulting meta-analysis uncovered extensive connections between Hsp90 and (1) general transcription, (2) ribosome biogenesis and (3) GTP binding proteins. Predictions from bioinformatic analyses were tested experimentally, supporting a role for Hsp90 in ribosome stability. Importantly, the integrated analysis of the 15°C heterozygous deletion pool screen with previously conducted 30°C and 37°C screens allows for essential genetic targets of Hsp90 to emerge. Altogether, these novel contributions enable a more complete picture of essential Hsp90 functions

Protein quality control: chaperones culling corrupt conformations

Achieving the correct balance between folding and degradation of misfolded proteins is critical for cell viability. The importance of defining the mechanisms and factors that mediate cytoplasmic quality control is underscored by the growing list of diseases associated with protein misfolding and aggregation. Molecular chaperones assist protein folding and also facilitate degradation of misfolded polypeptides by the ubiquitin-proteasome system. Here we discuss emerging links between folding and degradation machineries and highlight challenges for future research

The Hsp70 and TRiC/CCT Chaperone Systems Cooperate In Vivo To Assemble the Von Hippel-Lindau Tumor Suppressor Complex

The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex. Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70. In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC. We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC

Specific Molecular Chaperone Interactions and an ATP-dependent Conformational Change Are Required during Posttranslational Protein Translocation into the Yeast ER

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST–63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners