Abstract

Accumulation of aberrant proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response pathway that helps the cell to survive under these stress conditions. Herp is a mammalian ubiquitin domain protein, which is strongly induced by the unfolded protein response. It is involved in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We found that upon exposure of cells to ER stress, elevation of Herp steady state levels is accompanied by an enhanced association of Herp with pre-existing Hrd1. Hrd1-associated Herp is rapidly degraded and substituted by de novo synthesized Herp, suggesting a continuous turnover of the protein at Hrd1 complexes. Further analysis revealed the presence of multiple Hrd1 copies in a single complex enabling binding of a variable number of Herp molecules. Efficient ubiquitylation of the Hrd1-specific ERAD substrate α1-antitrypsin null Hong Kong (NHK) required the presence of the Herp UBL domain, which was also necessary for NHK degradation. In summary, we propose that binding of Herp to Hrd1-containing ERAD complexes positively regulates the ubiquitylation activity of these complexes, thus permitting survival of the cell during ER stress.

Highlights

  • The cytosolic surface of the endoplasmic reticulum (ER) membrane, which is a precondition for their extraction to the cytosol and their degradation by the 26 S proteasome [3]

  • Differential Regulation of Herp and Hrd1 Steady State Levels and Their Enhanced Association during ER Stress—Upon treatment of cells with ER stress-inducing agents such as thapsigargin and tunicamycin, both Herp and Hrd1 are induced by the UPR [4, 5, 17]

  • HeLa cells were treated with either thapsigargin or tunicamycin, and cell extracts were subjected to immunoprecipitation and analyzed by Western blotting

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Summary

Introduction

The cytosolic surface of the ER membrane, which is a precondition for their extraction to the cytosol and their degradation by the 26 S proteasome [3]. We show enhanced formation of dynamic HerpHrd1 complexes upon exposure of cells to ER stress and demonstrate the presence of oligomeric Hrd1 enabling binding of multiple Herp molecules. Differential Regulation of Herp and Hrd1 Steady State Levels and Their Enhanced Association during ER Stress—Upon treatment of cells with ER stress-inducing agents such as thapsigargin and tunicamycin, both Herp and Hrd1 are induced by the UPR [4, 5, 17].

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