Phospho-regulation of Cdc48/p97 cofactor protein Shp1/p47 in budding yeast

Shp1/p47 is a cofactor of the Cdc48/p97 complex involved in the energy-dependent segregation of intracellular aggregates and multiprotein complexes. We identified two serine residues (S108 and S315) in the S. Cerevisiae protein Shp1 which increase in phosphorylation upon various cell stresses such as heat shock, TORC1 inhibition, ER stress and oxidative stress. To identify binding partners of phosphorylated vs non-phosphorylated Shp1, we generated endogenous C-terminal 5xFLAG-tagged Shp1 yeast strains either harbouring either WT shp1 or CRISPR-modified S108A + S315A (2SA) shp1. These were cultured with 200 nM rapamycin treatment for 2 h to induce phosphorylation, prior to lysis, FLAG immunoprecipitation, peptide digestion and processing for LC-MS/MS.