Saccharomyces cerevisiae heat shock transcription factor regulates cell wall remodeling in response to heat shock

Abstract

The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates expression of genes encoding heat shock proteins and a variety of other proteins as well. To better understand the cellular roles of Hsf1, we screened multicopy suppressor genes of a temperature-sensitive hsf1 mutation. The RIM15 gene, encoding a protein kinase that is negatively regulated by the cyclic AMP-dependent protein kinase, was identified as a suppressor, but Rim15-regulated stress-responsive transcription factors, such as Msn2, Msn4, and Gis1, were unable to rescue the temperature-sensitive growth phenotype of the hsf1 mutant. Another class of suppressors encoded cell wall stress sensors, Wsc1, Wsc2, and Mid2, and the GDP/GTP exchange factor Rom2 that interacts with these cell wall sensors. Activation of a protein kinase, Pkc1, which is induced by these cell wall sensor proteins upon heat shock, but not activation of the Pkc1-regulated mitogen-activated protein kinase cascade, was necessary for the hsf1 suppression. Like Wsc-Pkc1 pathway mutants, hsf1 cells exhibited an osmotic remedial cell lysis phenotype at elevated temperatures. Several of the other suppressors were found to encode proteins functioning in cell wall organization. These results suggest that Hsf1 in concert with Pkc1 regulates cell wall remodeling in response to heat shock.

FIG. 1.

Characterization of multicopy suppressor genes. (A) Schematic diagram of structural motifs of Hsf1 and Hsf1 mutant constructs. The motifs indicated above Hsf1 are as follows: AR1 and AR2, activation domains; DBD, DNA-binding domain; oligomer, oligomerization domain; CE2, conserved element 2; CTM, C-terminal modulator. Numbers represent amino acid positions. Hsf1-Sp-CTMΔ contains amino acids 269 to 594 of S. pombe HSF but lacks the C-terminal 15 amino acids that function as a CTM domain. Hsf1-Hs contains amino acids 316 to 529 of human HSF1. (B) Multicopy suppression of growth defects at elevated temperature of hsf1-AR1Δ-ba1 cells. Wild-type HSF1 cells harboring empty vector (+ vector) and hsf1-AR1Δ-ba1 cells harboring empty vector or the vector bearing the suppressor genes (indicated by “+” and the gene name) were streaked on YPD medium and were incubated at 38°C for 2 days. The growth of hsf1-AR1Δ-ba1 cells harboring YEp-HSP82 is also shown. (C) RT-PCR analysis of multicopy suppressor gene effects on heat shock response target gene transcription. Wild-type HSF1 cells harboring empty vector (+ vector)and hsf1-ba1 cells harboring empty vector, YEp-RIM15, YEp-ROM2, YEp-RTS1, or YEp-ZDS1 were grown in ESD medium lacking uracil at 28°C, and then the temperature was shifted to 39°C. At the indicated times, aliquots of cells were removed and stored at −80°C. Total RNA prepared from each sample was subjected to RT-PCR analysis with primers for several heat-inducible genes (CUP1, CPR6, HSP42, and HSP78) and a control gene, ACT1.

FIG. 2.

Effect of components of the PKA pathway on the growth of hsf1-ba1 cells. (A) RT-PCR analysis of RIM15 overexpression effects on HSP12, HSP26, and SSA3 transcription. Wild-type HSF1 cells harboring empty vector (+ vector) and hsf1-ba1 cells harboring empty vector or YEp-RIM15 were grown in ESD medium lacking uracil, and total RNA prepared from each sample was subjected to RT-PCR analysis, as described for Fig. 1C. (B) Growth of hsf1-ba1 cells harboring multiple copies of the PKA pathway genes at elevated temperature. Wild-type HSF1 cells harboring empty vector (+ vector) and hsf1-ba1 cells harboring empty vector or the vector bearing various genes (indicated by “+” and the gene name) were streaked onto YPD medium and were incubated at 38°C for 2 days. (C) Growth of hsf1-ba1 cells containing mutations in the PKA pathway genes at elevated temperature. Wild-type HSF1 cells, their derivatives containing the indicated mutations, and mutant cells harboring YEp-RIM15 were streaked on YPD medium and were incubated at 38°C for 2 days.

FIG. 3.

Effect of components of the Wsc-Pkc1-Mpk1 pathway on the growth of hsf1-ba1 cells. (A) Suppression of temperature-sensitive growth defects of hsf1-ba1 cells by activation of the Wsc-Pkc1 pathway. Wild-type HSF1 cells harboring empty vector (+ vector) and hsf1-ba1 cells harboring empty vector, YEp-ROM2, YEp-PKC1, YCp-PKC1^R398P, YCp-BCK1-20, YCp-MKK1^S386P, YEp-MPK1, YEp-RLM1, or YEp-SWI4 were streaked on YPD medium and were incubated at 38°C for 2 days. (B) Heat-induced phosphorylation of Mpk1 in hsf1-ba1 cells. Wild-type HSF1 and hsf1-ba1 cells were grown in YPD medium at 28°C, and then the temperature was shifted to 39°C. At the indicated times, aliquots of cells were removed and protein extracts were prepared. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunoblot analysis with an antibody recognizing phosphorylated Mpk1.

FIG. 4.

Suppression of the lysis phenotype of hsf1-ba1 cells by osmotic stabilization. (A) Growth of cells containing hsf1-ba1 and wsc1Δ mutations under various conditions. Wild-type HSF1 cells (WT) and their derivatives containing hsf1-ba1 and wsc1Δ mutations were streaked onto YPD medium or YPD medium containing 1 M sorbitol and were incubated at 28, 35, or 38°C for 2 days. (B) Cell lysis assay of hsf1-ba1 and wsc1Δ cells grown at elevated temperature. Suspensions of the indicated cells were spotted on YPD medium or YPD medium containing 1 M sorbitol. Plates were incubated at 28°C for 1 day and then switched to 38°C and incubated overnight. The plate was overlaid with an alkaline phosphatase assay solution and incubated at 38°C for 1 h.

FIG. 5.

Growth of various hsf1 mutants under cell wall-stabilizing conditions. Cells expressing Hsf1 (WT), Hsf1-Sp-CTMΔ (Sp-CTMΔ), Hsf1-Hs (Hs), Hsf1-F256S (F256S), or Hsf1-N583 (N583) were streaked onto YPD medium or YPD containing 1 M sorbitol. Plates were incubated at 28 or at 37°C for 2 days. The lower left panel shows growth of the indicated hsf1 mutant cells harboring YEp-ROM2 on YPD medium at 37°C.