Limited-proteolysis coupled to mass spectrometry (LiP-MS) data on a serially filtered yeast lysate (the FLiP library) and on lysates of wild-type and Gcn5 mutant yeast strains grown under hydroxyurea stress and control conditions. Affinity purification mass spectrometry on Ada3 pulldowns from yeast grown under hydroxyurea stress and control conditions.

ABSTRACT: We applied serial ultrafiltration and limited proteolysis-coupled mass spectrometry (LiP-MS) to generate an atlas of peptides that act as markers for the formation or dissolution of protein-protein interactions (PPIs). Applied to Saccharomyces cerevisiae, the approach yielded 6,441 candidate PPI markers from 1,086 proteins, including 1,072 markers of known interfaces from 272 proteins. These include peptides mapping to both validated and putative protein binding interfaces, as well as markers of structural changes that accompany PPIs. The approach is based on detecting differences in protease susceptibility between the protein complex-bound and monomeric forms. We then used these peptide markers to track changes in protein-protein interactions in LiP-MS analyses of unfractionated lysates of yeast grown under hydroxyurea (HU) stress compared to control conditions. We captured known and novel protein complex rearrangements, supporting the key role played by Spt-Ada-Gcn5 acetyltransferase (SAGA) and the activity of its acetyltransferase Gcn5 in the stress response. We validated our observed rearrangement of SAGA with an AP-MS experiment comparing Ada3 pulldowns of yeast grown under HU and control conditions. We further examined how protein complex rearrangements change in Gcn5 mutant cells; we found a set of protein complexes that depend directly or indirectly on Gcn5 activity and identified a link between Gcn5 activity and the regulation of P-bodies.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Cathy Marulli  

LAB HEAD: Paola Picotti

PROVIDER: PXD055566 | Pride | 2024-10-17

REPOSITORIES: Pride