Glycogen Synthase Phosphatase Interacts with Heat Shock Factor To Activate CUP1 Gene Transcription in Saccharomyces cerevisiae

Abstract

Upon heat shock, transcription of many stress-inducible genes is rapidly and dramatically stimulated by heat shock factor (HSF). A central region of the yeast HSF (designated HSFrr for “repression region”) was previously identified and proposed to be involved in repressing the activation domain under non-heat-shock conditions. Here, we used the phage display system to isolate proteins that interact with HSFrr. This should identify factors that modulate HSF activity or directly participate in HSF-mediated transcriptional activation. We constructed a randomly sheared yeast genomic library to express yeast proteins on the surface of λ phage. HSFrr binding phages were selected by cycles of affinity chromatography. DNA sequencing identified an HSFrr-interacting phage that contains the GAC1 gene. The GAC1 gene encodes the regulatory subunit for a type 1 serine/threonine phosphoprotein phosphatase, Glc7. Both gac1 and glc7 mutations had little effect on HSF activation of gene transcription of two heat shock genes,SSA4 and HSP82. In contrast, heat shock induction of CUP1 gene expression was completely abolished in a glc7 mutant and reduced in a gac1 mutant. The results demonstrate that the Glc7 phosphatase and its Gac1 regulatory subunit play positive roles in HSF activation of CUP1 transcription.

ACKNOWLEDGMENTS

We are especially grateful to R. Hoess for generating the starting vector to generate the λ phage library and for advice about conducting the phage display selection. We are grateful to K. Tatchell for the gift of strains, antibody, and plasmid and for communication of results prior to publication. We thank D. Thiele for freely exchanging information. We thank K. Leptos for constructing plasmid pKCL002. J. T. Lin thanks P. Mason for advice on the pull-down assay and E. Guzmán for assisting with the production of the photographic figures. We thank the members of the Lis laboratory for general interest and helpful discussions. We thank P. Mason and D. Lee for their comments on the manuscript.

This work was supported by Public Health Service grant GM25232 (awarded to J. T. Lis) from the National Institutes of Health. J. T. Lin was supported by NIH postdoctoral fellowship award 1F32GM17989-01.