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Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in Saccharomyces cerevisiae.

Kumar N, Gaur D, Gupta A, Puri A, Sharma D - PLoS Genet. (2015)

Bottom Line: We show that Cpr7 interacts with Ure2 and enhances its fibrillation.The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [PSI+], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions.Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.

View Article: PubMed Central - PubMed

Affiliation: Council of Scientific and Industrial Research-Institute of Microbial Technology, Chandigarh, India.

ABSTRACT
The role of Hsp70 chaperones in yeast prion propagation is well established. Highly conserved Hsp90 chaperones participate in a number of cellular processes, such as client protein maturation, protein degradation, cellular signalling and apoptosis, but little is known about their role in propagation of infectious prion like aggregates. Here, we examine the influence of Hsp90 in the maintenance of yeast prion [URE3] which is a prion form of native protein Ure2, and reveal a previously unknown role of Hsp90 as an important regulator of [URE3] stability. We show that the C-terminal MEEVD pentapeptide motif, but not the client maturation activity of Hsp90, is essential for [URE3] prion stability. In testing deletions of various Hsp90 co-chaperones known to bind this motif, we find the immunophilin homolog Cpr7 is essential for [URE3] propagation. We show that Cpr7 interacts with Ure2 and enhances its fibrillation. The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [PSI+], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions. Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.

No MeSH data available.


Related in: MedlinePlus

Cpr7 is required for [URE3] stability.(A) Effect of deleting genes encoding Hsp90 co-chaperones on prion propagation. Multiple single-knockout strains constructed as described in Materials and Methods were streaked onto a ½ YPD plate and [URE3] was monitored after 3 days of incubation at 30°C and 2 days at room temperature. As seen, the deletion of gene encoding Cpr7 leads to [ure-o] phenotype. (B) Cells were grown in YPAD liquid medium, further grown from O.D.600nm = 0.02 to 1.7 and then plated on ½ YPD. The colonies were then replica-plated onto ½ YPD and adenine deficient plates. As seen, though SY187 [URE3] cells grow normally on adenine deficient medium, only about 2–3% of colonies from cpr7Δ strain grew on solid medium lacking adenine. (C) The effect of Hsp90 co-chaperone deletion on [PSI+] propagation. Deletion strains of the ade2-1 background were assessed as in panel (A). (D) [URE3] diploid strain heterozygous for cpr7Δ (CPR7/cpr7::KanMX) was sporulated and dissected. [URE3] phenotype segregated 2:2 and all cpr7Δ (G418 resistant) spores were found to be [ure-o] as seen by red colony color phenotype.
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pgen.1005567.g002: Cpr7 is required for [URE3] stability.(A) Effect of deleting genes encoding Hsp90 co-chaperones on prion propagation. Multiple single-knockout strains constructed as described in Materials and Methods were streaked onto a ½ YPD plate and [URE3] was monitored after 3 days of incubation at 30°C and 2 days at room temperature. As seen, the deletion of gene encoding Cpr7 leads to [ure-o] phenotype. (B) Cells were grown in YPAD liquid medium, further grown from O.D.600nm = 0.02 to 1.7 and then plated on ½ YPD. The colonies were then replica-plated onto ½ YPD and adenine deficient plates. As seen, though SY187 [URE3] cells grow normally on adenine deficient medium, only about 2–3% of colonies from cpr7Δ strain grew on solid medium lacking adenine. (C) The effect of Hsp90 co-chaperone deletion on [PSI+] propagation. Deletion strains of the ade2-1 background were assessed as in panel (A). (D) [URE3] diploid strain heterozygous for cpr7Δ (CPR7/cpr7::KanMX) was sporulated and dissected. [URE3] phenotype segregated 2:2 and all cpr7Δ (G418 resistant) spores were found to be [ure-o] as seen by red colony color phenotype.

Mentions: As compromising Hsp90 client maturation activity does not affect [URE3], the appearance of [ure-o] cells in the His6-Hsp82ΔMEEVD strain points toward a crucial role of Hsp90 co-chaperones in prion propagation. In order to examine the role of various Hsp90 co-factors, we created many single-knockout S. cerevisiae strains each lacking an Hsp90 co-chaperone (Sti1, Sba1, Cpr6, Cpr7, Ppt1, Tah1, Hch1 or Aha1) and monitored [URE3]. As shown in Fig 2A the deletion of non-TPR Hsp90 co-chaperones (Sba1, Hch1 or Aha1) had no effect on the prion propagation. Among the TPR domain containing proteins, only deletion of the gene encoding Cpr7 profoundly affects [URE3] stability as seen by the appearance of red colony color as well as poor growth on solid medium lacking adenine (Fig 2A and 2B). To examine whether this effect was [URE3] specific, we further constructed similar single-knockouts in strain 779-6A propagating the other well studied yeast prion [PSI+]. In contrast to its requirement for [URE3], lack of Cpr7 had no effect on [PSI+] stability which is also in agreement with a previously reported study [50]. Similarly, other knockout strains carrying a single gene deletion encoding one of seven other Hsp90 co-chaperones also show no apparent differences in [PSI+] stability (Fig 2C). We also examined the effect of deleting Cpr7 on strong and weak [PSI+] variants and found no difference in the prion stability in the presence and absence of Cpr7 (S3 Fig). Thus both weak and strong [PSI+] variants remain largely unaffected by Cpr7 deletion which is distinct from the effect of Hsp70 co-chaperone Sse1 where deletion of Sse1 destabilize weak [PSI+] but has no effect on strong [PSI+] prions [51].


Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in Saccharomyces cerevisiae.

Kumar N, Gaur D, Gupta A, Puri A, Sharma D - PLoS Genet. (2015)

Cpr7 is required for [URE3] stability.(A) Effect of deleting genes encoding Hsp90 co-chaperones on prion propagation. Multiple single-knockout strains constructed as described in Materials and Methods were streaked onto a ½ YPD plate and [URE3] was monitored after 3 days of incubation at 30°C and 2 days at room temperature. As seen, the deletion of gene encoding Cpr7 leads to [ure-o] phenotype. (B) Cells were grown in YPAD liquid medium, further grown from O.D.600nm = 0.02 to 1.7 and then plated on ½ YPD. The colonies were then replica-plated onto ½ YPD and adenine deficient plates. As seen, though SY187 [URE3] cells grow normally on adenine deficient medium, only about 2–3% of colonies from cpr7Δ strain grew on solid medium lacking adenine. (C) The effect of Hsp90 co-chaperone deletion on [PSI+] propagation. Deletion strains of the ade2-1 background were assessed as in panel (A). (D) [URE3] diploid strain heterozygous for cpr7Δ (CPR7/cpr7::KanMX) was sporulated and dissected. [URE3] phenotype segregated 2:2 and all cpr7Δ (G418 resistant) spores were found to be [ure-o] as seen by red colony color phenotype.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608684&req=5

pgen.1005567.g002: Cpr7 is required for [URE3] stability.(A) Effect of deleting genes encoding Hsp90 co-chaperones on prion propagation. Multiple single-knockout strains constructed as described in Materials and Methods were streaked onto a ½ YPD plate and [URE3] was monitored after 3 days of incubation at 30°C and 2 days at room temperature. As seen, the deletion of gene encoding Cpr7 leads to [ure-o] phenotype. (B) Cells were grown in YPAD liquid medium, further grown from O.D.600nm = 0.02 to 1.7 and then plated on ½ YPD. The colonies were then replica-plated onto ½ YPD and adenine deficient plates. As seen, though SY187 [URE3] cells grow normally on adenine deficient medium, only about 2–3% of colonies from cpr7Δ strain grew on solid medium lacking adenine. (C) The effect of Hsp90 co-chaperone deletion on [PSI+] propagation. Deletion strains of the ade2-1 background were assessed as in panel (A). (D) [URE3] diploid strain heterozygous for cpr7Δ (CPR7/cpr7::KanMX) was sporulated and dissected. [URE3] phenotype segregated 2:2 and all cpr7Δ (G418 resistant) spores were found to be [ure-o] as seen by red colony color phenotype.
Mentions: As compromising Hsp90 client maturation activity does not affect [URE3], the appearance of [ure-o] cells in the His6-Hsp82ΔMEEVD strain points toward a crucial role of Hsp90 co-chaperones in prion propagation. In order to examine the role of various Hsp90 co-factors, we created many single-knockout S. cerevisiae strains each lacking an Hsp90 co-chaperone (Sti1, Sba1, Cpr6, Cpr7, Ppt1, Tah1, Hch1 or Aha1) and monitored [URE3]. As shown in Fig 2A the deletion of non-TPR Hsp90 co-chaperones (Sba1, Hch1 or Aha1) had no effect on the prion propagation. Among the TPR domain containing proteins, only deletion of the gene encoding Cpr7 profoundly affects [URE3] stability as seen by the appearance of red colony color as well as poor growth on solid medium lacking adenine (Fig 2A and 2B). To examine whether this effect was [URE3] specific, we further constructed similar single-knockouts in strain 779-6A propagating the other well studied yeast prion [PSI+]. In contrast to its requirement for [URE3], lack of Cpr7 had no effect on [PSI+] stability which is also in agreement with a previously reported study [50]. Similarly, other knockout strains carrying a single gene deletion encoding one of seven other Hsp90 co-chaperones also show no apparent differences in [PSI+] stability (Fig 2C). We also examined the effect of deleting Cpr7 on strong and weak [PSI+] variants and found no difference in the prion stability in the presence and absence of Cpr7 (S3 Fig). Thus both weak and strong [PSI+] variants remain largely unaffected by Cpr7 deletion which is distinct from the effect of Hsp70 co-chaperone Sse1 where deletion of Sse1 destabilize weak [PSI+] but has no effect on strong [PSI+] prions [51].

Bottom Line: We show that Cpr7 interacts with Ure2 and enhances its fibrillation.The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [PSI+], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions.Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.

View Article: PubMed Central - PubMed

Affiliation: Council of Scientific and Industrial Research-Institute of Microbial Technology, Chandigarh, India.

ABSTRACT
The role of Hsp70 chaperones in yeast prion propagation is well established. Highly conserved Hsp90 chaperones participate in a number of cellular processes, such as client protein maturation, protein degradation, cellular signalling and apoptosis, but little is known about their role in propagation of infectious prion like aggregates. Here, we examine the influence of Hsp90 in the maintenance of yeast prion [URE3] which is a prion form of native protein Ure2, and reveal a previously unknown role of Hsp90 as an important regulator of [URE3] stability. We show that the C-terminal MEEVD pentapeptide motif, but not the client maturation activity of Hsp90, is essential for [URE3] prion stability. In testing deletions of various Hsp90 co-chaperones known to bind this motif, we find the immunophilin homolog Cpr7 is essential for [URE3] propagation. We show that Cpr7 interacts with Ure2 and enhances its fibrillation. The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [PSI+], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions. Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.

No MeSH data available.


Related in: MedlinePlus