Cns1 Is an Essential Protein Associated with the Hsp90 Chaperone Complex in Saccharomyces cerevisiae That Can Restore Cyclophilin 40-Dependent Functions in cpr7ΔCells

ABSTRACT

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 cpr7Δ slow-growth phenotype. The screen identified a single gene, designatedCNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Δ growth defect. Overexpression ofCNS1 in cpr7Δ 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.

ACKNOWLEDGMENTS

We thank S. Lindquist for plasmids, S. cerevisiaestrains, and anti-Hsp82 antibodies, D. Toft for anti-Sti1 antibodies, and E. Craig for anti-Ssa antibodies. We thank D. Winge for the pHSE2-lacZ and pHSE12-lacZ reporter genes.

J.A.M. was supported in part by the ARCS Foundations and by predoctoral biotechnology training grant T32GM-08449. H.M.K. was supported by a National Science Foundation Research Experiences for Undergraduates supplement to R.F.G. This work was supported by National Science Foundation grant MCB-9724050 to R.F.G.

ADDENDUM IN PROOF

Our two-hybrid results with Cns1 are consistent with the findings of Young et al. (J. Biol. Chem. 273:18007–18010, 1998) published during review of this paper, in which the 104 carboxy-terminal residues of Hsp90 were sufficient to interact with TPR-containing Hsp90-associated proteins FKBP51, FKBP52, and hTom34p.