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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 enhances in vitro fibrillization of Ure2.Purified Ure2 (48 μM) was incubated at 37°C with and without Cpr6, Cpr7 or 7TPR (30 μM each) and 500μM ThioflavinT. The ThT fluorescence was monitored at 485nm upon excitation at 450nm. As seen, ThT fluorescence intensity was further enhanced upon incubation of Ure2 with Cpr7 or 7TPR.
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pgen.1005567.g007: Cpr7 enhances in vitro fibrillization of Ure2.Purified Ure2 (48 μM) was incubated at 37°C with and without Cpr6, Cpr7 or 7TPR (30 μM each) and 500μM ThioflavinT. The ThT fluorescence was monitored at 485nm upon excitation at 450nm. As seen, ThT fluorescence intensity was further enhanced upon incubation of Ure2 with Cpr7 or 7TPR.

Mentions: As Cpr7 interacts with Ure2 and is required for stable [URE3] propagation, we speculated that Cpr7 might have the ability to modulate Ure2 fibrillation required for stable [URE3] maintenance. In order to examine the effect of Cpr7 on amyloid forming tendency of Ure2, we monitored fibrillation of Ure2 in vitro using Thioflavin T (ThT), a dye that forms fluorescent complexes with amyloids, in the presence and absence of Cpr7. As expected ThT fluorescence intensity increases upon incubation with Ure2 at 37°C, suggesting spontaneous fibrillation of Ure2 with a lag of about 2–3 min. Cpr7 and Cpr6 alone did not affect ThT fluorescence intensity. Pre-incubation of Ure2 with Cpr7 did not affect the lag time. However, the rate of amyloid formation and amyloid yield increased significantly, indicating that Cpr7 promotes Ure2 fibrillation. The increase in Ure2 fibrillation was observed only upon incubation with Cpr7 and not its near homolog Cpr6 (Fig 7). The increase in fluorescence intensity was not due to a direct effect of Cpr7 on ThT as fluorescence intensity was similar when similar amounts of pre-aggregated Ure2 protein, with and without Cpr7, was measured (S7A and S7B Fig).


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 enhances in vitro fibrillization of Ure2.Purified Ure2 (48 μM) was incubated at 37°C with and without Cpr6, Cpr7 or 7TPR (30 μM each) and 500μM ThioflavinT. The ThT fluorescence was monitored at 485nm upon excitation at 450nm. As seen, ThT fluorescence intensity was further enhanced upon incubation of Ure2 with Cpr7 or 7TPR.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005567.g007: Cpr7 enhances in vitro fibrillization of Ure2.Purified Ure2 (48 μM) was incubated at 37°C with and without Cpr6, Cpr7 or 7TPR (30 μM each) and 500μM ThioflavinT. The ThT fluorescence was monitored at 485nm upon excitation at 450nm. As seen, ThT fluorescence intensity was further enhanced upon incubation of Ure2 with Cpr7 or 7TPR.
Mentions: As Cpr7 interacts with Ure2 and is required for stable [URE3] propagation, we speculated that Cpr7 might have the ability to modulate Ure2 fibrillation required for stable [URE3] maintenance. In order to examine the effect of Cpr7 on amyloid forming tendency of Ure2, we monitored fibrillation of Ure2 in vitro using Thioflavin T (ThT), a dye that forms fluorescent complexes with amyloids, in the presence and absence of Cpr7. As expected ThT fluorescence intensity increases upon incubation with Ure2 at 37°C, suggesting spontaneous fibrillation of Ure2 with a lag of about 2–3 min. Cpr7 and Cpr6 alone did not affect ThT fluorescence intensity. Pre-incubation of Ure2 with Cpr7 did not affect the lag time. However, the rate of amyloid formation and amyloid yield increased significantly, indicating that Cpr7 promotes Ure2 fibrillation. The increase in Ure2 fibrillation was observed only upon incubation with Cpr7 and not its near homolog Cpr6 (Fig 7). The increase in fluorescence intensity was not due to a direct effect of Cpr7 on ThT as fluorescence intensity was similar when similar amounts of pre-aggregated Ure2 protein, with and without Cpr7, was measured (S7A and S7B Fig).

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