Reference: Flynn JM, et al. (2024) Dominant negative mutations in yeast Hsp90 indicate triage decision mechanism targeting client proteins for degradation. Mol Biol Cell mbcE24070309

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Abstract


Dominant negative mutations provide valuable tools for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative (DN) mutations in yeast Hsp90. In a previous mutational scan of the ATPase domain of Hsp90, we noticed that many mutations were at very low frequency after outgrowth in cells co-expressing WT Hsp90. Most of these depleted variants were located at the hinge of a lid that closes over ATP. To quantify toxic effects in the hinge regions, we performed mutational scanning using an inducible promoter and identified 113 variants with strong toxic effects. We analyzed individual DN mutations in detail and found that addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. FRET assays performed on individual DN mutants indicate the linkage between ATPase activity and formation of the closed structure is disrupted. DN Hsp90 decreased the expression level of two model Hsp90 clients, glucocorticoid receptor (GR) and v-src kinase. Using MG132, we found that GR was rapidly destabilized in a proteasome-dependent fashion. Biochemical analyses indicate that ATP hydrolysis by Hsp90 from open conformations can lead to ubiquitin-dependent client degradation.

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Journal Article
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Flynn JM, Joyce ME, Bolon DNA
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