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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 interacts with Ure2.The purified His6-Cpr7 was adsorbed onto Cobalt metal affinity resin and further incubated with yeast lysate expressing either Hsp82 or Hsp82ΔMEEVD as sole Hsp90. Upon washing, the fraction bound to His6-Cpr7 was eluted with 25mM EDTA, and analyzed for Ure2 and Hsp90 using anti-Ure2 and anti-Hsp90 antibodies, respectively. His6-Cpr7 was probed with anti-His6 antibody.
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pgen.1005567.g006: Cpr7 interacts with Ure2.The purified His6-Cpr7 was adsorbed onto Cobalt metal affinity resin and further incubated with yeast lysate expressing either Hsp82 or Hsp82ΔMEEVD as sole Hsp90. Upon washing, the fraction bound to His6-Cpr7 was eluted with 25mM EDTA, and analyzed for Ure2 and Hsp90 using anti-Ure2 and anti-Hsp90 antibodies, respectively. His6-Cpr7 was probed with anti-His6 antibody.

Mentions: Cpr7 belongs to the family of immunophilins that are also known to interact with many unfolded substrates to prevent their aggregation and keep them in a folding competent state [54] [56]. We used a pull-down assay using purified His6-Cpr7 as bait to examine interaction of Cpr7 with Ure2. The cell lysate from wild type [ure-o] cells was fractionated and the supernatant was incubated with Cpr7-bound cobalt based metal affinity resin. The unbound proteins were washed and the bound fraction was probed by immunobloting with antibodies specific against Ure2 or Hsp90. In agreement with previous studies, Hsp90 was identified in fractions bound to His6-Cpr7 (Fig 6). The deletion of MEEVD leads to a significant decrease in Hsp82 interaction with Cpr7. Interestingly, Ure2 was also detected in the fraction that bound to His6-Cpr7. The presence of Ure2 in this fraction suggests that Ure2 either interacts with Cpr7 or Hsp82. We thus examined Ure2 interaction with Cpr7 using cell lysate from cells expressing Hsp82ΔMEEVD. If the presence of Ure2 in the fraction bound to Cpr7 is indirectly due to its interaction with Hsp82, the loss of Cpr7-Hsp82 interaction upon deletion of C-terminus MEEVD motif of Hsp82 would lead to a decrease in Ure2 level in the bound fraction. As shown, however, a similar level of Ure2 was detected in the bound fraction obtained using lysates from cells expressing either Hsp82 or Hsp82ΔMEEVD, suggesting that Cpr7 interacts directly with Ure2.


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 interacts with Ure2.The purified His6-Cpr7 was adsorbed onto Cobalt metal affinity resin and further incubated with yeast lysate expressing either Hsp82 or Hsp82ΔMEEVD as sole Hsp90. Upon washing, the fraction bound to His6-Cpr7 was eluted with 25mM EDTA, and analyzed for Ure2 and Hsp90 using anti-Ure2 and anti-Hsp90 antibodies, respectively. His6-Cpr7 was probed with anti-His6 antibody.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005567.g006: Cpr7 interacts with Ure2.The purified His6-Cpr7 was adsorbed onto Cobalt metal affinity resin and further incubated with yeast lysate expressing either Hsp82 or Hsp82ΔMEEVD as sole Hsp90. Upon washing, the fraction bound to His6-Cpr7 was eluted with 25mM EDTA, and analyzed for Ure2 and Hsp90 using anti-Ure2 and anti-Hsp90 antibodies, respectively. His6-Cpr7 was probed with anti-His6 antibody.
Mentions: Cpr7 belongs to the family of immunophilins that are also known to interact with many unfolded substrates to prevent their aggregation and keep them in a folding competent state [54] [56]. We used a pull-down assay using purified His6-Cpr7 as bait to examine interaction of Cpr7 with Ure2. The cell lysate from wild type [ure-o] cells was fractionated and the supernatant was incubated with Cpr7-bound cobalt based metal affinity resin. The unbound proteins were washed and the bound fraction was probed by immunobloting with antibodies specific against Ure2 or Hsp90. In agreement with previous studies, Hsp90 was identified in fractions bound to His6-Cpr7 (Fig 6). The deletion of MEEVD leads to a significant decrease in Hsp82 interaction with Cpr7. Interestingly, Ure2 was also detected in the fraction that bound to His6-Cpr7. The presence of Ure2 in this fraction suggests that Ure2 either interacts with Cpr7 or Hsp82. We thus examined Ure2 interaction with Cpr7 using cell lysate from cells expressing Hsp82ΔMEEVD. If the presence of Ure2 in the fraction bound to Cpr7 is indirectly due to its interaction with Hsp82, the loss of Cpr7-Hsp82 interaction upon deletion of C-terminus MEEVD motif of Hsp82 would lead to a decrease in Ure2 level in the bound fraction. As shown, however, a similar level of Ure2 was detected in the bound fraction obtained using lysates from cells expressing either Hsp82 or Hsp82ΔMEEVD, suggesting that Cpr7 interacts directly with Ure2.

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