The Hsp110 protein chaperone Sse1 is required for yeast cell wall integrity and morphogenesis

Abstract

Molecular chaperones direct refolding and triage decisions and support signal transduction responses to cytotoxic stress. The eukaryotic chaperone Hsp110 is represented by the SSE1/2 genes in Saccharomyces cerevisiae, which act as nucleotide exchange factors (NEFs) for cognate cytosolic Hsp70 chaperones. In this report, we present evidence that Sse1 is required for signaling through the cell integrity pathway via partnership with Hsp90 and the terminal MAP kinase Slt2. We found that sse1Delta and sti1Delta mutant cells share the typical cell integrity mutant phenotypes of osmoremediated temperature-sensitive growth and sensitivity to cell wall-damaging agents. Sse1 binds to Slt2 in vivo and similar to Hsp90 mutants, Slt2 stability and phosphorylation is not compromised in sse1Delta cells, whereas activation of the downstream transcription factor Rlm1 is abolished. In addition to Rlm1, Slt2 activates the Swi4/Swi6 heterodimer SBF in response to cell wall damage. SSE1 displayed dramatic synthetic phenotypes when disrupted in combination with mutations in SBF and the related Mbp1/Swi6 heterodimer MBF, characterized by severe growth and morphological defects. These defects were reversed by restoration of Hsp70 NEF activity, providing a mechanistic model wherein Sse1 functionally partners with Hsp90 as an Hsp70 NEF to promote client protein maturation and interaction with downstream effectors.

Fig. 1

SSE1 and STI1 mutants exhibit defects in cell integrity. a Serial dilutions of wild type (DS10) and the indicated isogenic knockouts, sse1Δ (DTY249) and sti1Δ (CN11), were spotted onto YPD, YPD containing 1 M sorbitol, or YPD containing 0.4 M NaCl plates and incubated at 30, 37, or 39°C for 2 days. A single equivalent dilution representing approximately 10³ cells is shown for each condition. b A strain containing the ssa1-45 allele as the sole copy of Ssa Hsp70 and its isogenic SSA1 wild type parent were serially diluted and grown at 30 or 37°C on YPD plates containing 1 M sorbitol or 0.4 M NaCl as described in (a). c Serial dilutions of wild type (BY4741) and the isogenic sse1Δ disruption (LSY4) containing the indicated alleles were spotted onto YPD, YPD plus caffeine, or YPD plus Congo Red and incubated at 30°C for 2 days. A single dilution is shown as in (a)

Fig. 2

Sse1 physically interacts with Slt2 and is required for its activation. a FLAG immunoprecipitations were performed on protein extracts derived from W303 cells harboring p416GPD-FLAG-SLT2 or p416GPD as described in “Materials and methods.” Immunoblot analysis using anti-Sse1 and anti-FLAG antibodies was carried out on the extracts and immunoprecipitates. The extract lanes represent 5% of the total amount of extract used for the immunoprecipitation. b A strain carrying an integrated 13×-myc-tagged SLT2 allele (wild type, BY2207) and the isogenic SSE1 deletion (sse1Δ, LSY19) were grown to mid-log phase at 25°C followed by shifting of the cultures to 37°C and removal of aliquots at 0, 30, and 60 min postshift. Protein extracts were prepared followed by anti-myc and anti-phospho-Slt2 immunoblot analysis. c Wild type (DS10), sse1Δ (KMY69), swi4Δ (LSY1), and rlm1Δ (LSY2) cells were transformed with the PST1-lacZ reporter plasmid to measure transcriptional activation by Rlm1 and β-galactosidase activity assayed before and after a 1.5 h heat shock at 37°C

Fig. 3

Synthetic growth and morphology defects between SSE1 and SBF components. a Wild type (BY4741), and the indicated isogenic derivative strains (see Table 1) were serially diluted, plated on YPD and incubated at 30 and 37°C for 2 days. A parallel dilution series was subject to the alkaline phosphatase cell leakage assay after 2 days of growth at 37°C as described in “Materials and methods,” and a representative dilution spot is shown for all strains (BCIP). b The same strains were analyzed for morphology using Normarski microscopy. Representative micrographs are shown. c Quantitation of the morphological types observed in (b). Populations of at least 100 cells were categorized as normal (white bars), elongated (gray bars), or amorphous (black bars) as described in “Materials and methods”

Fig. 4

Hsp70 nucleotide exchange factor activity is required for normal growth and morphology in swi6Δ mutants. sse1Δ swi6Δ cells were transformed with the indicated plasmids bearing SSE1 alleles, SSE2 or FES1 (see text for explanation), and morphological defects assessed as in Fig. 3. Populations of at least 200 cells for each strain were categorized as normal (white bars), elongated (gray bars), or amorphous (black bars) as described in “Materials and methods”

Fig. 5

SSE1 genetically interacts with RLM1 and MBF in addition to SBF. Wild type (BY4741) and the indicated isogenic derivative strains (see Table 1) were serially diluted, spotted onto YPD plates and incubated at 30°C for 2 days

Fig. 6

A multichaperone complex supports cell integrity signaling. A multichaperone complex that includes at least Hsp90, Hsp70 (Ssa), Hsp110 (Sse), and Stil interacts with the terminal Slt2 MAP kinase and is required for proper signaling to the effector transcription factors SBF and Rlm1. SBF and Rlm1 differentially regulate genes involved in cell wall repair and morphogenesis. Chaperone components may also be required for Slt2-independent functions of MBF