Hop/Sti1 phosphorylation inhibits its co-chaperone function

Area of Science:

• Molecular biology
• Cellular biology
• Protein biochemistry

Background:

• Molecular chaperones Hsp90 and Hsp70 are crucial for protein homeostasis in eukaryotes.
• The co-chaperone Sti1/Hop facilitates the interaction between Hsp90 and Hsp70, enabling client protein transfer and regulation.
• Understanding the conserved and divergent roles of Sti1/Hop across species is essential for comprehending chaperone network dynamics.

Purpose of the Study:

• To investigate the conserved functions of yeast Sti1 and human Hop co-chaperones.
• To explore the impact of phosphorylation on the regulatory mechanisms of Sti1 and Hop.
• To determine how species-specific phosphorylation affects the interaction of Hop/Sti1 with Hsp90 and Hsp70 and client activation.

Main Methods:

• Biochemical assays to assess simultaneous binding of Hsp90 and Hsp70.
• Enzyme activity assays to measure inhibition of Hsp90 ATPase activity.
• In vivo studies using yeast and human cell models to evaluate client activation.
• Site-directed mutagenesis to create phospho-mimetic variants of Sti1/Hop.
• Analysis of protein-protein interactions and structural changes upon phosphorylation.

Main Results:

• Yeast Sti1 and human Hop share conserved functions, including binding both Hsp90 and Hsp70, inhibiting Hsp90 ATPase activity, and supporting client activation.
• Both Sti1 and Hop are regulated by inhibitory phosphorylation, but the specific sites and effects are species-specific.
• Phospho-mimetic variants exhibit reduced client activation and altered Hsp70 binding affinity.
• Human Hop is more stringently regulated, with phosphorylation affecting Hsp90 interaction and inducing structural changes.

Conclusions:

• The fundamental roles of Sti1/Hop in linking Hsp90 and Hsp70 are conserved across yeast and humans.
• Phosphorylation serves as a critical regulatory mechanism for Sti1/Hop, with distinct species-specific outcomes.
• Differential regulation of Hop by phosphorylation impacts its interaction network and structural integrity, highlighting evolutionary divergence in chaperone regulation.