PgmNr Y3195: Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperone-dependent regulation of ribonucleotide reductase.

Authors:
Andrew Truman 1 ; Laura Knighton 1 ; Isaac Sluder 1 ; Donald Wolfgeher 2 ; Stephen Kron 2


Institutes
1) University of North Carolina at Charlotte, Charlotte, NC; 2) University of Chicago, Chicago, IL 60637.


Keyword: Proteomics

Abstract:

The highly conserved molecular chaperones Hsp90 and Hsp70 are indispensible for folding and maturation of a significant fraction of the proteome, including many proteins involved in signal transduction and stress response. To examine the dynamics of chaperone-client interactions after DNA damage, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to characterize interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate (MMS). Of 256 proteins identified and quantified via 16O/18O labeling and LC-MS/MS, 142 are novel Hsp70/90 interactors. Nearly all interactions remained unchanged or decreased after DNA damage, but 5 proteins increased interactions with Ssa1 and/or Hsp82, including the ribonucleotide reductase (RNR) subunit Rnr4. Inhibiting Hsp70 or 90 chaperone activity destabilized Rnr4 in yeast and its vertebrate homolog hRMM2 in breast cancer cells. In turn, pre-treatment of cancer cells with chaperone inhibitors sensitized cells to the RNR inhibitor gemcitabine, suggesting a novel chemotherapy strategy.



Yeast Database Genetic Index
1. gene symbol: Ssa1; systematic name: YAL005C
2. gene symbol: Hsp82; systematic name: YPL240C
3. gene symbol: Rnr2; systematic name: YJL026W
4. gene symbol: Rnr4; systematic name: YGR180C