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Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
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Dominant negative mutations in yeast Hsp90 indicate triage decision mechanism targeting client proteins for

Julia M Flynn1, Margot E Joyce1, Daniel N A Bolon1

  • 1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605.

Molecular Biology of the Cell
|November 20, 2024
PubMed

Insights

Dominant negative mutations in yeast Hsp90 were identified. These mutations disrupt protein structure and client protein degradation, offering insights into Hsp90 mechanisms.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Dominant negative (DN) mutations are crucial for studying protein function but challenging to isolate due to toxicity.
  • Hsp90 is a molecular chaperone involved in protein folding and stability.

Purpose of the Study:

  • To identify and characterize dominant negative (DN) mutations in yeast Hsp90.
  • To investigate the impact of these mutations on Hsp90's ATPase activity, structure, and client protein regulation.

Main Methods:

  • Mutational scanning using an inducible promoter to identify toxic variants.
  • Analysis of individual DN mutations, including effects of preventing ATP hydrolysis (E33A mutation).
  • Förster Resonance Energy Transfer (FRET) assays to assess structural changes.
  • Assays measuring client protein expression levels (glucocorticoid receptor, v-src kinase) and degradation pathways (proteasome, MG132).

Main Results:

  • Identified 113 variants with strong toxic effects, primarily in the Hsp90 hinge region.
  • The E33A mutation abrogated the DN phenotype, indicating the importance of ATP hydrolysis.
  • FRET assays revealed disrupted linkage between ATPase activity and Hsp90's closed conformation.
  • DN Hsp90 reduced expression of client proteins (GR, v-src) and promoted their proteasome-dependent degradation.

Conclusions:

  • Dominant negative mutations in yeast Hsp90 disrupt its ATPase activity and structural dynamics.
  • These mutations lead to client protein destabilization and degradation, providing insights into Hsp90's role in protein homeostasis.
  • The findings highlight the critical link between Hsp90's ATPase cycle and its chaperone function.

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