Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1.

ABSTRACT:

Background

Endoplasmic reticulum (ER)-associated degradation (ERAD) regulates protein homeostasis in the secretory pathway by targeting misfolded or unassembled proteins for degradation by the proteasome. Hrd1 is a conserved multi-spanning membrane bound ubiquitin ligase required for ubiquitination of many aberrant ER proteins, but few endogenous substrates of Hrd1 have been identified to date.

Methods

Using a SILAC-based quantitative proteomic approach combined with CRISPR-mediated gene silencing, we searched for endogenous physiological substrates of Hrd1. We used RNA microarray, immunoblotting, cycloheximide chase combined with chemical genetics to define the role of Hrd1 in regulating the stability of endogenous ERAD substrates.

Results

We identified 58 proteins w

SUBMITTER: Ye Y 

PROVIDER: S-EPMC6103995 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature