The heat shock response is a highly conserved mechanism that allows cells to withstand a variety of stress conditions. Activation of this response is characterized by increased synthesis of heat shock proteins (HSPs), which protect cellular proteins from stress-induced denaturation. Heat shock transcription factors (HSFs) are required for increased expression of HSPs during stress conditions and can be found in complexes containing components of the Hsp90 molecular chaperone machinery, raising the possibility that Hsp90 is involved in regulation of the heat shock response. To test this, we have assessed the effects of mutations that impair activity of the Hsp90 machinery on heat shock related events in Saccharomyces cerevisiae. Mutations that either reduce the level of Hsp90 protein or eliminate Cpr7, a CyP-40-type cyclophilin required for full Hsp90 function, resulted in increased HSF-dependent activities. Genetic tests also revealed that Hsp90 and Cpr7 function synergistically to repress gene expression from HSFdependent promoters. Conditional loss of Hsp90 activity resulted in both increased HSF-dependent gene expression and acquisition of a thermotolerant phenotype. Our results reveal that Hsp90 and Cpr7 are required for negative regulation of the heat shock response under both stress and nonstress conditions and establish a specific endogenous role for the Hsp90 machinery in S. cerevisiae.All cells possess a defense mechanism known as the heat shock response, which allows them to survive exposure to otherwise lethal doses of certain stresses (1-3). These stresses include environmental challenges, such as elevated temperatures, and pathophysiological states, such as viral infections (4). The heat shock response is characterized by increased synthesis of a set of proteins collectively referred to as heat shock proteins (HSPs) 1 whose principal role is to assist target substrates in their synthesis, transport, and proper folding (5-7). The requirement for chaperoning activities increases as cells are exposed to elevated temperatures or to other conditions that promote protein denaturation and aggregation. Because chaperoning activity is also crucial for the function of proteins not exposed to stress, HSPs play important roles for life under normal conditions as well.Expression of HSPs is under the control of heat shock transcription factors (HSFs) (4,8,9). In most eukaryotic systems, HSF is maintained as a monomer unable to bind DNA until activated by stress (4,8). Activated HSF forms homotrimers capable of binding to heat shock elements (HSEs) present at promoters of genes encoding HSPs, ultimately leading to transcriptional activation (4,8). The monomer to trimer transition is believed to be negatively regulated, at least in part, by Hsp70 (4,8). The acquisition of transcriptional activity by HSF is correlated with increased phosphorylation (10); however, the functional relationship between phosphorylation and regulation of the heat shock response is still not fully understood. In contrast to most eukaryoti...

Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60 v-src . In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7⌬ slow-growth phenotype. The screen identified a single gene, designated CNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7⌬ growth defect. Overexpression of CNS1 in cpr7⌬ cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.Cells rely on molecular chaperones to facilitate the folding of nascent polypeptides, prevent protein aggregation, and enable proper inter-and intramolecular interactions. Indispensable in both Saccharomyces cerevisiae (5) and Drosophila melanogaster (13), Hsp90 is one of the most abundant and highly conserved chaperones. In vitro, Hsp90 displays general chaperone properties by preventing the aggregation of proteins such as citrate synthase (52) and casein kinase II (30) and by maintaining ␤-galactosidase (21) in a folding-competent state. In vivo, however, Hsp90 is not required for the folding of most proteins (33) but instead plays a key role in the maturation of a small subset of proteins typically involved in signal transduction (3,32,42). It is needed for the maturation of certain steroid hormone receptors (40,43,44), basic helix-loop-helix transcription factors (45

We describe a one-step gene replacement method based on fusion PCR that can be used to mutagenize essential genes at their endogenous locus. Marker-fusion PCR can facilitate transfer of alleles between strains as well as PCR-based techniques, such as site-directed and error-prone PCR mutagenesis, all without cloning or strain constructions. With this method, PCR is used to fuse a mutagenized fragment to an overlapping fragment containing a selectable marker flanked by regions of homology to the target. By transforming yeast with these PCR products, specific mutations are introduced at the endogenous locus through homologous recombination. We tested the 'marker-fusion PCR' method using the budding yeast CDC28 gene and were able to efficiently introduce site-directed mutations and integrate genomic or plasmid-borne mutant alleles. As a further application for this method, we used a spiked oligonucleotide to randomize the coding sequence for a single domain of CDC28 and were able to construct highly mutagenized libraries for this region.