Mutational analysis of the Hsp70 substrate-binding domain: Correlating molecular-level changes with in vivo function
Linan Xu1, Hong Zhang2, Daragh D Cuskelly1
1Department of Biology, Maynooth University, Maynooth, Ireland.
Molecular Microbiology
|December 20, 2020
Summary
Mutations in heat shock protein 70 (Hsp70) disrupt its function, impairing cellular stress response and prion propagation in yeast. The size of a specific amino acid side chain is crucial for Hsp70 stability and function.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Heat shock protein 70 (Hsp70) is a conserved chaperone essential for protein homeostasis.
- Mutations in the substrate-binding domain (SBD) of Hsp70 impair its function, affecting cellular stress responses and prion propagation.
- The β6/β7 region of the SBD is critical for Hsp70 stability and function.
Purpose of the Study:
- To elucidate the mechanisms of Hsp70 loss of function caused by SBD disruption.
- To investigate the role of key residues in the β6/β7 region of the Hsp70 SBD.
- To understand how mutations affect Hsp70 stability, ATPase activity, and cellular function.
Main Methods:
- Targeted mutational analysis of key residues (F475 and 483) in the Hsp70 SBD β6/β7 region.
- Assessment of SBD stability, cleavage, and degradation pathways (vacuolar vs. proteasomal).
- Measurement of Hsp70 ATPase activity and in vivo protein re-folding capacity.
Main Results:
- The hydrophobic side chain size at residue 475 is critical for Hsp70 SBD stability and function.
- Mutations at residues 475 and 483 lead to SBD instability, cleavage, and degradation.
- Hsp70-Ssa cleavage is mediated by a vacuolar carboxypeptidase (Pep4)-dependent mechanism.
- Mutations compromise ATPase activity, reduce protein re-folding, and deplete cytosolic Hsp70 levels.
- Mutated yeast cells exhibit impaired stress response and inability to propagate the [PSI+] prion.
Conclusions:
- The F475 residue and the size of its hydrophobic side chain are essential for Hsp70 stability and function.
- SBD disruption leads to Hsp70 degradation via a Pep4-dependent pathway.
- Compromised Hsp70 function due to mutations affects cellular stress tolerance and prion propagation in yeast.
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