Direct involvement of Hsp70 ATP hydrolysis in Ubr1-dependent quality control

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• Chaperones play a dual role in protein folding and degradation, a process termed protein triage.
• Understanding protein triage is vital for both fundamental science and therapeutic applications.
• In yeast, misfolded proteins are ubiquitinated by E3 ligases Ubr1 and San1, facilitating protein triage studies.

Purpose of the Study:

• To investigate the direct involvement of chaperones in Ubr1-mediated ubiquitination.
• To elucidate the specific requirements of the HSP70 chaperone system in protein triage.
• To develop an in vitro system for studying chaperone-dependent protein degradation.

Main Methods:

• Development of a bead-based assay with immobilized, releasable misfolded protein.
• In vitro ubiquitination assays using purified components.
• Analysis of the role of HSP70, its ATPase cycle, and cochaperones in Ubr1-mediated ubiquitination.

Main Results:

• HSP70, its ATPase cycle, and cochaperones are essential for Ubr1-mediated ubiquitination.
• An in vitro assay confirmed that only HSP70 and its associated components were required for ubiquitination.
• The study identified specific requirements for HSP70's ATPase cycle, Sse1's nucleotide exchange, and J-proteins in Ubr1-mediated quality control.

Conclusions:

• The HSP70 ATPase cycle is a key determinant in directing proteins for ubiquitination and degradation.
• This suggests a model where efficiently folded proteins are spared, while misfolded proteins are tagged for degradation.
• The findings provide insights into the molecular mechanisms of cellular protein quality control and triage.