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The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly.

ABSTRACT: Sgt1p is a well-conserved protein proposed to be involved in a number of cellular processes. Genetic studies of budding yeast suggest a role for SGT1 in signal transduction, cell cycle advance, and chromosome segregation. Recent evidence has linked Sgt1p to HSP90 chaperones, although the precise relationship between these proteins is unclear. To further explore the role of Sgt1p in these processes, we have characterized the interactions among Sgt1p, the inner kinetochore complex CBF3, and HSP90 chaperones. We show that the amino terminus of Sgt1p interacts with CBF3 subunits Skp1p and Ctf13p. HSP90 interacts with Sgt1p and, in combination with the carboxy terminus of Sgt1p, regulates the interaction between Sgt1p and Skp1p in a nucleotide-dependent manner. While the Sgt1p-Skp1p interaction is required for CBF3 assembly, mutations that stabilize this interaction prevent the turnover of protein complexes important for CBF3 assembly. We propose that HSP90 and Sgt1p act together as a molecular switch, maintaining transient interactions required to balance protein complex assembly with turnover.

SUBMITTER: Lingelbach LB

PROVIDER: S-EPMC517876 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly.

Lingelbach Linda B LB Kaplan Kenneth B KB

Molecular and cellular biology 20041001 20

Sgt1p is a well-conserved protein proposed to be involved in a number of cellular processes. Genetic studies of budding yeast suggest a role for SGT1 in signal transduction, cell cycle advance, and chromosome segregation. Recent evidence has linked Sgt1p to HSP90 chaperones, although the precise relationship between these proteins is unclear. To further explore the role of Sgt1p in these processes, we have characterized the interactions among Sgt1p, the inner kinetochore complex CBF3, and HSP90 ...[more]

PMID: 15456868

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Project description:Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398-719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1-550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310-529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.

Dataset Information

The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly.

Publications

The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly.

Similar Datasets

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