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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 tetratricopeptide domain is important for [URE3] prion propagation.(A) Schematic of domain architecture of Cpr6, Cpr7 and their hybrid proteins. (B) Strains expressing Cpr6, Cpr7 or hybrids in cpr7Δ genetic background were constructed and monitored for [URE3] as described in Fig 1A. ½ YPD plates were further replicated onto SD lacking histidine (SD, -His) or adenine (SD, -Ade). Shown is the growth after 1day (1D) or 2 days (2D) of incubation at 30°C after replica plating from ½ YPD plate. As seen by white colony color phenotype cells expressing Cpr7, 6PPI/7TPR or 7TPR support stable [URE3]. Red colony color phenotype is seen only in those colonies that either lost the transformed plasmid (due to growth on ½ YPD), harbour empty plasmid or the plasmid encoding for Cpr6, 7PPI/6TPR.
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pgen.1005567.g004: Cpr7 tetratricopeptide domain is important for [URE3] prion propagation.(A) Schematic of domain architecture of Cpr6, Cpr7 and their hybrid proteins. (B) Strains expressing Cpr6, Cpr7 or hybrids in cpr7Δ genetic background were constructed and monitored for [URE3] as described in Fig 1A. ½ YPD plates were further replicated onto SD lacking histidine (SD, -His) or adenine (SD, -Ade). Shown is the growth after 1day (1D) or 2 days (2D) of incubation at 30°C after replica plating from ½ YPD plate. As seen by white colony color phenotype cells expressing Cpr7, 6PPI/7TPR or 7TPR support stable [URE3]. Red colony color phenotype is seen only in those colonies that either lost the transformed plasmid (due to growth on ½ YPD), harbour empty plasmid or the plasmid encoding for Cpr6, 7PPI/6TPR.

Mentions: The Hsp90 associated immunophilins Cpr6 and Cpr7 belong to the PPIase family of enzymes and share more than 50% sequence similarity, yet they can act in distinct cellular processes [53] [54]. In order to examine whether Cpr6 can complement the role of Cpr7 with regard to [URE3], the cpr7Δ strain harbouring Cpr7 on a URA3-based plasmid (pRS316PCPR7-CPR7) was transformed with a plasmid encoding Cpr6 or Cpr7 (pRS413PTEF-CPR6 or pRS413PTEF-CPR7, respectively) regulated by the TEF promoter. We then used FOA selection to isolate cells having lost the resident URA3 plasmid. The resulting cpr7Δ strains with plasmids encoding Cpr6 or Cpr7 were grown in liquid medium selecting for plasmid maintenance and then spread onto ½ YPD plates. Colony color was noted after incubation at 30°C for 3–4 days, at which time the ½ YPD plates were replicated onto SD medium lacking histidine (for monitoring plasmid loss) or adenine (for monitoring [URE3]). As shown in Fig 4B, cells expressing Cpr7 colonies were white on ½ YPD and grew well on medium lacking adenine, suggesting that the plasmid expressed Cpr7 functionally complements the chromosomal knockout of CPR7 with regard to [URE3]. In contrast, cells harbouring either an empty plasmid (ev) or a plasmid encoding Cpr6 show similar red colony color on ½ YPD. After continued incubation on adenine deficient growth medium at 30°C, cells expressing Cpr6 grew only poorly, which is similar to cells with the empty plasmid. Thus, Cpr6 was unable to complement Cpr7 function with regard to [URE3] maintenance.


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 tetratricopeptide domain is important for [URE3] prion propagation.(A) Schematic of domain architecture of Cpr6, Cpr7 and their hybrid proteins. (B) Strains expressing Cpr6, Cpr7 or hybrids in cpr7Δ genetic background were constructed and monitored for [URE3] as described in Fig 1A. ½ YPD plates were further replicated onto SD lacking histidine (SD, -His) or adenine (SD, -Ade). Shown is the growth after 1day (1D) or 2 days (2D) of incubation at 30°C after replica plating from ½ YPD plate. As seen by white colony color phenotype cells expressing Cpr7, 6PPI/7TPR or 7TPR support stable [URE3]. Red colony color phenotype is seen only in those colonies that either lost the transformed plasmid (due to growth on ½ YPD), harbour empty plasmid or the plasmid encoding for Cpr6, 7PPI/6TPR.
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getmorefigures.php?uid=PMC4608684&req=5

pgen.1005567.g004: Cpr7 tetratricopeptide domain is important for [URE3] prion propagation.(A) Schematic of domain architecture of Cpr6, Cpr7 and their hybrid proteins. (B) Strains expressing Cpr6, Cpr7 or hybrids in cpr7Δ genetic background were constructed and monitored for [URE3] as described in Fig 1A. ½ YPD plates were further replicated onto SD lacking histidine (SD, -His) or adenine (SD, -Ade). Shown is the growth after 1day (1D) or 2 days (2D) of incubation at 30°C after replica plating from ½ YPD plate. As seen by white colony color phenotype cells expressing Cpr7, 6PPI/7TPR or 7TPR support stable [URE3]. Red colony color phenotype is seen only in those colonies that either lost the transformed plasmid (due to growth on ½ YPD), harbour empty plasmid or the plasmid encoding for Cpr6, 7PPI/6TPR.
Mentions: The Hsp90 associated immunophilins Cpr6 and Cpr7 belong to the PPIase family of enzymes and share more than 50% sequence similarity, yet they can act in distinct cellular processes [53] [54]. In order to examine whether Cpr6 can complement the role of Cpr7 with regard to [URE3], the cpr7Δ strain harbouring Cpr7 on a URA3-based plasmid (pRS316PCPR7-CPR7) was transformed with a plasmid encoding Cpr6 or Cpr7 (pRS413PTEF-CPR6 or pRS413PTEF-CPR7, respectively) regulated by the TEF promoter. We then used FOA selection to isolate cells having lost the resident URA3 plasmid. The resulting cpr7Δ strains with plasmids encoding Cpr6 or Cpr7 were grown in liquid medium selecting for plasmid maintenance and then spread onto ½ YPD plates. Colony color was noted after incubation at 30°C for 3–4 days, at which time the ½ YPD plates were replicated onto SD medium lacking histidine (for monitoring plasmid loss) or adenine (for monitoring [URE3]). As shown in Fig 4B, cells expressing Cpr7 colonies were white on ½ YPD and grew well on medium lacking adenine, suggesting that the plasmid expressed Cpr7 functionally complements the chromosomal knockout of CPR7 with regard to [URE3]. In contrast, cells harbouring either an empty plasmid (ev) or a plasmid encoding Cpr6 show similar red colony color on ½ YPD. After continued incubation on adenine deficient growth medium at 30°C, cells expressing Cpr6 grew only poorly, which is similar to cells with the empty plasmid. Thus, Cpr6 was unable to complement Cpr7 function with regard to [URE3] maintenance.

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