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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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Spore wall construction defects in the hsp82-I578F mutant. (A) D818 cells carrying pHsp82-GFP as the sole copy of Hsp90 exhibit normal spore formation as shown by transmission electron microscopy. (B–D) D818 cells carrying pHsp82-I578F-GFP as the sole copy of Hsp90 exhibit diverse sporulation defects. Inset in B is a 2× digital zoom of dashed rectangle. Bar, 2 μm.
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Figure 6: Spore wall construction defects in the hsp82-I578F mutant. (A) D818 cells carrying pHsp82-GFP as the sole copy of Hsp90 exhibit normal spore formation as shown by transmission electron microscopy. (B–D) D818 cells carrying pHsp82-I578F-GFP as the sole copy of Hsp90 exhibit diverse sporulation defects. Inset in B is a 2× digital zoom of dashed rectangle. Bar, 2 μm.

Mentions: Similar to metazoan differentiation programs, spore formation is induced in response to a nutrient stimulus in specific cell types and is characterized by the ordered progression of morphogenic stages that lead to a differentiated state (Neiman, 2005). The spore wall is composed of two inner spore wall layers, which resemble a vegetative cell, and two outer layers containing spore-specific materials, chitosan and dityrosine (Neiman, 2005). To examine the structural and morphological defects associated with loss of Hsp90 nuclear accumulation at high resolution, sporulated HSP82-GFP and hsp82-I578F-GFP diploid cells were examined by transmission electron microscopy. The typical ultrastructure of a wild-type spore is exemplified by the ascus shown in Figure 6A. In contrast, hsp82-I578F-GFP asci exhibited multiple defects in spore wall construction. A possible defect in assembly of the outer dityrosine layer is exhibited in Figure 6B, where the spore wall seems to have ruptured, releasing its contents but retaining an incomplete dityrosine ghost structure. Figure 6, C and D, illustrates asci with partial to complete failure of spore assembly, resulting in catastrophic sporulation. Nuclear-localized Hsp90 may therefore be required for one or more critical steps in spore morphogenesis, including but not limited to spore wall construction.


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

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

Spore wall construction defects in the hsp82-I578F mutant. (A) D818 cells carrying pHsp82-GFP as the sole copy of Hsp90 exhibit normal spore formation as shown by transmission electron microscopy. (B–D) D818 cells carrying pHsp82-I578F-GFP as the sole copy of Hsp90 exhibit diverse sporulation defects. Inset in B is a 2× digital zoom of dashed rectangle. Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801720&req=5

Figure 6: Spore wall construction defects in the hsp82-I578F mutant. (A) D818 cells carrying pHsp82-GFP as the sole copy of Hsp90 exhibit normal spore formation as shown by transmission electron microscopy. (B–D) D818 cells carrying pHsp82-I578F-GFP as the sole copy of Hsp90 exhibit diverse sporulation defects. Inset in B is a 2× digital zoom of dashed rectangle. Bar, 2 μm.
Mentions: Similar to metazoan differentiation programs, spore formation is induced in response to a nutrient stimulus in specific cell types and is characterized by the ordered progression of morphogenic stages that lead to a differentiated state (Neiman, 2005). The spore wall is composed of two inner spore wall layers, which resemble a vegetative cell, and two outer layers containing spore-specific materials, chitosan and dityrosine (Neiman, 2005). To examine the structural and morphological defects associated with loss of Hsp90 nuclear accumulation at high resolution, sporulated HSP82-GFP and hsp82-I578F-GFP diploid cells were examined by transmission electron microscopy. The typical ultrastructure of a wild-type spore is exemplified by the ascus shown in Figure 6A. In contrast, hsp82-I578F-GFP asci exhibited multiple defects in spore wall construction. A possible defect in assembly of the outer dityrosine layer is exhibited in Figure 6B, where the spore wall seems to have ruptured, releasing its contents but retaining an incomplete dityrosine ghost structure. Figure 6, C and D, illustrates asci with partial to complete failure of spore assembly, resulting in catastrophic sporulation. Nuclear-localized Hsp90 may therefore be required for one or more critical steps in spore morphogenesis, including but not limited to spore wall construction.

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