Analysis of p62-UBA interacting proteins

Abstract

Sequestosome 1/p62 interacts with and traffics K63-polyubiquitinated proteins through its ubiquitin associating domain (UBA). Herein we demonstrate that the hyperaccumulation of K63-polyubiquitinated proteins occurs in the absence of p62 making knock-out mice a rich source of K63-polyubiquitinated proteins for proteomic analysis. Formic acid fraction of wild-type and knock-out mice brain were subjected to p62 GST-UBA pull down, then shotgun LC-MS/MS was employed to identify those p62 UBA-interacting proteins. Using this approach, we identified 30 proteins consisting of nine classes: cytoskeleton / structural protein, energy / metabolism, membrane transport / ion channel, signaling, chaperon, intracellular trafficking, nuclear, neurogenesis, and unknown / unassigned proteins. The results of Western-blotting and immunoprecipitation of a subset reveal that those p62-interacting proteins are accumulated in p62 knock-out mice brains and are K63-polyubiquitinated. Our results support a model whereby p62 shuttles K63-polyubiquitinated proteins for proteasomal degradation.