Limits...
Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.

Zhao R, Kakihara Y, Gribun A, Huen J, Yang G, Khanna M, Costanzo M, Brost RL, Boone C, Hughes TR, Yip CM, Houry WA - J. Cell Biol. (2008)

Bottom Line: Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein.As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions.Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

Show MeSH
Mapping the interactions between Hsp90, Tah1, and Pih1. (A) Schematic representation of the domains in Tah1, Pih1, Sti1, and Hsp82. (B) His6-tagged proteins were incubated with untagged proteins, and complexes were then isolated using Ni–nitrilotriacetic acid resin, resolved on SDS-PAGE gels, and stained with silver stain (left) or Coomassie Brilliant blue (right). (C) The interaction between Hsp82-C and Pih1 was characterized using yeast 2H. The 2H analysis was performed in WT diploid cells (top) or cells deleted of TAH1 (middle). The bottom panel shows control cells containing pOBD2 empty vector and pOAD2-Pih1. Cells were grown in selection medium without histidine. (D) Interactions between Hsp82, Tah1, and Pih1 were analyzed by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (E) A protein interaction model for Hsp82, Tah1, and Pih1. N, N-terminal domain of Hsp82; M, middle domain of Hsp82; C, C-terminal domain of Hsp82.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2234237&req=5

fig1: Mapping the interactions between Hsp90, Tah1, and Pih1. (A) Schematic representation of the domains in Tah1, Pih1, Sti1, and Hsp82. (B) His6-tagged proteins were incubated with untagged proteins, and complexes were then isolated using Ni–nitrilotriacetic acid resin, resolved on SDS-PAGE gels, and stained with silver stain (left) or Coomassie Brilliant blue (right). (C) The interaction between Hsp82-C and Pih1 was characterized using yeast 2H. The 2H analysis was performed in WT diploid cells (top) or cells deleted of TAH1 (middle). The bottom panel shows control cells containing pOBD2 empty vector and pOAD2-Pih1. Cells were grown in selection medium without histidine. (D) Interactions between Hsp82, Tah1, and Pih1 were analyzed by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (E) A protein interaction model for Hsp82, Tah1, and Pih1. N, N-terminal domain of Hsp82; M, middle domain of Hsp82; C, C-terminal domain of Hsp82.

Mentions: In the Saccharomyces cerevisiae (yeast) cytoplasm, there are two virtually identical isoforms of Hsp90: one termed Hsp82, which is heat shock induced, and the other is termed Hsc82, which is constitutively expressed. Structural studies, sequence conservation, and proteolysis analyses of Hsp90s have indicated the presence of at least three domains (Fig. 1 A). The N-terminal domain is the site of ATP binding and hydrolysis and is connected to a middle domain. The middle domain has been proposed to be the site of cochaperone binding. Finally, the C-terminal domain provides a strong dimerization interface (for review seePearl and Prodromou, 2006). In this paper, the term Hsp90 is used when we do not explicitly distinguish between Hsp82 and Hsc82.


Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.

Zhao R, Kakihara Y, Gribun A, Huen J, Yang G, Khanna M, Costanzo M, Brost RL, Boone C, Hughes TR, Yip CM, Houry WA - J. Cell Biol. (2008)

Mapping the interactions between Hsp90, Tah1, and Pih1. (A) Schematic representation of the domains in Tah1, Pih1, Sti1, and Hsp82. (B) His6-tagged proteins were incubated with untagged proteins, and complexes were then isolated using Ni–nitrilotriacetic acid resin, resolved on SDS-PAGE gels, and stained with silver stain (left) or Coomassie Brilliant blue (right). (C) The interaction between Hsp82-C and Pih1 was characterized using yeast 2H. The 2H analysis was performed in WT diploid cells (top) or cells deleted of TAH1 (middle). The bottom panel shows control cells containing pOBD2 empty vector and pOAD2-Pih1. Cells were grown in selection medium without histidine. (D) Interactions between Hsp82, Tah1, and Pih1 were analyzed by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (E) A protein interaction model for Hsp82, Tah1, and Pih1. N, N-terminal domain of Hsp82; M, middle domain of Hsp82; C, C-terminal domain of Hsp82.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234237&req=5

fig1: Mapping the interactions between Hsp90, Tah1, and Pih1. (A) Schematic representation of the domains in Tah1, Pih1, Sti1, and Hsp82. (B) His6-tagged proteins were incubated with untagged proteins, and complexes were then isolated using Ni–nitrilotriacetic acid resin, resolved on SDS-PAGE gels, and stained with silver stain (left) or Coomassie Brilliant blue (right). (C) The interaction between Hsp82-C and Pih1 was characterized using yeast 2H. The 2H analysis was performed in WT diploid cells (top) or cells deleted of TAH1 (middle). The bottom panel shows control cells containing pOBD2 empty vector and pOAD2-Pih1. Cells were grown in selection medium without histidine. (D) Interactions between Hsp82, Tah1, and Pih1 were analyzed by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (E) A protein interaction model for Hsp82, Tah1, and Pih1. N, N-terminal domain of Hsp82; M, middle domain of Hsp82; C, C-terminal domain of Hsp82.
Mentions: In the Saccharomyces cerevisiae (yeast) cytoplasm, there are two virtually identical isoforms of Hsp90: one termed Hsp82, which is heat shock induced, and the other is termed Hsc82, which is constitutively expressed. Structural studies, sequence conservation, and proteolysis analyses of Hsp90s have indicated the presence of at least three domains (Fig. 1 A). The N-terminal domain is the site of ATP binding and hydrolysis and is connected to a middle domain. The middle domain has been proposed to be the site of cochaperone binding. Finally, the C-terminal domain provides a strong dimerization interface (for review seePearl and Prodromou, 2006). In this paper, the term Hsp90 is used when we do not explicitly distinguish between Hsp82 and Hsc82.

Bottom Line: Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein.As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions.Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

Show MeSH