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Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

Tapia H, Morano KA - Mol. Biol. Cell (2009)

Bottom Line: Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation.The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele.Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX 77030, USA.

ABSTRACT
The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

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Reduction in Hsp90 activity is sufficient to cause defective sporulation and is coincident with loss of nuclear accumulation. (A) D818 cells bearing pHsp82-GFP or pHsp82-I578F-GFP, or plasmids pTCA-Hsp82, pTCA-Hsp82/G313N and pTCA-Hsp82/E431K and p416Gal-v-Src were induced by growth in the presence of galactose for 10 h (gal), or maintained in glucose (glc), and protein extracts were assayed by immunoblot for bulk tyrosine phosphorylation (α-p-tyr). Levels of glucose phosphate dehydrogenase were assessed as a gel loading control (α-GPD). (B) D818 cells bearing the plasmids pTCA-Hsp82, pTCA-Hsp82/G313N, and pTCA-Hsp82/E431K were sporulated and efficiency calculated as in Figure 5. (C) Wild-type diploid cells were treated with the indicated concentrations of macbecin during sporulation and efficiency calculated as in Figure 5. Error bars represent the SD from two replicates. (D) D818 cells bearing the pTCA-Hsp82-yEm-RFP, pTCA-Hsp82/G313N-yEm-RFP and pTCA-Hsp82/E431K-yEm-RFP plasmids (see Materials and Methods) were grown to quiescent phase and subcellular distribution assessed.
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Figure 7: Reduction in Hsp90 activity is sufficient to cause defective sporulation and is coincident with loss of nuclear accumulation. (A) D818 cells bearing pHsp82-GFP or pHsp82-I578F-GFP, or plasmids pTCA-Hsp82, pTCA-Hsp82/G313N and pTCA-Hsp82/E431K and p416Gal-v-Src were induced by growth in the presence of galactose for 10 h (gal), or maintained in glucose (glc), and protein extracts were assayed by immunoblot for bulk tyrosine phosphorylation (α-p-tyr). Levels of glucose phosphate dehydrogenase were assessed as a gel loading control (α-GPD). (B) D818 cells bearing the plasmids pTCA-Hsp82, pTCA-Hsp82/G313N, and pTCA-Hsp82/E431K were sporulated and efficiency calculated as in Figure 5. (C) Wild-type diploid cells were treated with the indicated concentrations of macbecin during sporulation and efficiency calculated as in Figure 5. Error bars represent the SD from two replicates. (D) D818 cells bearing the pTCA-Hsp82-yEm-RFP, pTCA-Hsp82/G313N-yEm-RFP and pTCA-Hsp82/E431K-yEm-RFP plasmids (see Materials and Methods) were grown to quiescent phase and subcellular distribution assessed.

Mentions: The preceding results raise the possibility that functional Hsp90 is required in the nucleus before initiation of sporulation to generate intact, viable spores. An alternative explanation is that the hsp82-I578F allele, in addition to blocking nuclear translocation, is hypomorphic for Hsp90 activity and that loss of chaperone function is sufficient to cause the defects we have observed. We tested these two scenarios by assaying the ability of hsp82-I578F to support activation and function of the mammalian Hsp90 client tyrosine kinase v-Src in yeast (Xu and Lindquist, 1993). When expressed from a galactose-inducible promoter, v-Src promiscuously phosphorylates several unknown endogenous yeast proteins, detectable by immunoblot with anti-phosphotyrosine antibodies (Xu and Lindquist, 1993). As shown in Figure 7A, the characteristic banding pattern was observed in cells bearing wild-type HSP82 and the v-Src plasmid grown in galactose. No signal was observed in glucose medium, confirming the specificity of the antibody. However hsp82-I578F cells exhibited substantial reduction in tyrosine phosphorylation, indicative of reduced Hsp90 chaperone activity. To place these results in context, we also examined two previously characterized HSP82 mutant alleles, hsp82-G313N and hsp82-E431K (Bohen and Yamamoto, 1993). When compared directly with a strain bearing the appropriate HSP82 wild-type allele (see Materials and Methods), hsp82-G313N displayed complete loss of v-Src activity, whereas hsp82-E431K seemed normal (Figure 7A). These results are consistent with a previous functional analysis using steroid receptor clients, demonstrating that the G313N substitution results in severe diminution of Hsp90 activity, whereas E431K behaves essentially as wild type with the exception of glucocorticoid signaling (Bohen and Yamamoto, 1993; Bohen, 1995). We next investigated the ability of the G313N and E431K mutants to support sporulation as the sole source of Hsp90 in diploid cells. Strikingly, whereas hsp82-E431K and the corresponding wild-type diploids exhibited similar sporulation, the hsp82-G313N cells displayed a dramatic sporulation defect in line with that observed with the nonlocalizing hsp82-I578F mutant (Figure 7B). These findings suggest that reduction in Hsp90 chaperone activity is sufficient to inhibit proper sporulation. To further test this hypothesis, we asked whether pharmacological ablation of endogenous Hsp90 in wild-type cells with the Hsp90-specific inhibitor macbecin would have the same effect (Whitesell et al., 1994). Indeed, as shown in Figure 7C, macbecin treatment resulted in dose-dependent inhibition of overall sporulation efficiency, completely blocking sporulation at established concentrations (∼20 μM) known to inhibit Hsp90 activities in yeast cells (Bohen, 1998).


Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

Tapia H, Morano KA - Mol. Biol. Cell (2009)

Reduction in Hsp90 activity is sufficient to cause defective sporulation and is coincident with loss of nuclear accumulation. (A) D818 cells bearing pHsp82-GFP or pHsp82-I578F-GFP, or plasmids pTCA-Hsp82, pTCA-Hsp82/G313N and pTCA-Hsp82/E431K and p416Gal-v-Src were induced by growth in the presence of galactose for 10 h (gal), or maintained in glucose (glc), and protein extracts were assayed by immunoblot for bulk tyrosine phosphorylation (α-p-tyr). Levels of glucose phosphate dehydrogenase were assessed as a gel loading control (α-GPD). (B) D818 cells bearing the plasmids pTCA-Hsp82, pTCA-Hsp82/G313N, and pTCA-Hsp82/E431K were sporulated and efficiency calculated as in Figure 5. (C) Wild-type diploid cells were treated with the indicated concentrations of macbecin during sporulation and efficiency calculated as in Figure 5. Error bars represent the SD from two replicates. (D) D818 cells bearing the pTCA-Hsp82-yEm-RFP, pTCA-Hsp82/G313N-yEm-RFP and pTCA-Hsp82/E431K-yEm-RFP plasmids (see Materials and Methods) were grown to quiescent phase and subcellular distribution assessed.
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Figure 7: Reduction in Hsp90 activity is sufficient to cause defective sporulation and is coincident with loss of nuclear accumulation. (A) D818 cells bearing pHsp82-GFP or pHsp82-I578F-GFP, or plasmids pTCA-Hsp82, pTCA-Hsp82/G313N and pTCA-Hsp82/E431K and p416Gal-v-Src were induced by growth in the presence of galactose for 10 h (gal), or maintained in glucose (glc), and protein extracts were assayed by immunoblot for bulk tyrosine phosphorylation (α-p-tyr). Levels of glucose phosphate dehydrogenase were assessed as a gel loading control (α-GPD). (B) D818 cells bearing the plasmids pTCA-Hsp82, pTCA-Hsp82/G313N, and pTCA-Hsp82/E431K were sporulated and efficiency calculated as in Figure 5. (C) Wild-type diploid cells were treated with the indicated concentrations of macbecin during sporulation and efficiency calculated as in Figure 5. Error bars represent the SD from two replicates. (D) D818 cells bearing the pTCA-Hsp82-yEm-RFP, pTCA-Hsp82/G313N-yEm-RFP and pTCA-Hsp82/E431K-yEm-RFP plasmids (see Materials and Methods) were grown to quiescent phase and subcellular distribution assessed.
Mentions: The preceding results raise the possibility that functional Hsp90 is required in the nucleus before initiation of sporulation to generate intact, viable spores. An alternative explanation is that the hsp82-I578F allele, in addition to blocking nuclear translocation, is hypomorphic for Hsp90 activity and that loss of chaperone function is sufficient to cause the defects we have observed. We tested these two scenarios by assaying the ability of hsp82-I578F to support activation and function of the mammalian Hsp90 client tyrosine kinase v-Src in yeast (Xu and Lindquist, 1993). When expressed from a galactose-inducible promoter, v-Src promiscuously phosphorylates several unknown endogenous yeast proteins, detectable by immunoblot with anti-phosphotyrosine antibodies (Xu and Lindquist, 1993). As shown in Figure 7A, the characteristic banding pattern was observed in cells bearing wild-type HSP82 and the v-Src plasmid grown in galactose. No signal was observed in glucose medium, confirming the specificity of the antibody. However hsp82-I578F cells exhibited substantial reduction in tyrosine phosphorylation, indicative of reduced Hsp90 chaperone activity. To place these results in context, we also examined two previously characterized HSP82 mutant alleles, hsp82-G313N and hsp82-E431K (Bohen and Yamamoto, 1993). When compared directly with a strain bearing the appropriate HSP82 wild-type allele (see Materials and Methods), hsp82-G313N displayed complete loss of v-Src activity, whereas hsp82-E431K seemed normal (Figure 7A). These results are consistent with a previous functional analysis using steroid receptor clients, demonstrating that the G313N substitution results in severe diminution of Hsp90 activity, whereas E431K behaves essentially as wild type with the exception of glucocorticoid signaling (Bohen and Yamamoto, 1993; Bohen, 1995). We next investigated the ability of the G313N and E431K mutants to support sporulation as the sole source of Hsp90 in diploid cells. Strikingly, whereas hsp82-E431K and the corresponding wild-type diploids exhibited similar sporulation, the hsp82-G313N cells displayed a dramatic sporulation defect in line with that observed with the nonlocalizing hsp82-I578F mutant (Figure 7B). These findings suggest that reduction in Hsp90 chaperone activity is sufficient to inhibit proper sporulation. To further test this hypothesis, we asked whether pharmacological ablation of endogenous Hsp90 in wild-type cells with the Hsp90-specific inhibitor macbecin would have the same effect (Whitesell et al., 1994). Indeed, as shown in Figure 7C, macbecin treatment resulted in dose-dependent inhibition of overall sporulation efficiency, completely blocking sporulation at established concentrations (∼20 μM) known to inhibit Hsp90 activities in yeast cells (Bohen, 1998).

Bottom Line: Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation.The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele.Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX 77030, USA.

ABSTRACT
The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

Show MeSH
Related in: MedlinePlus