A Proteasomal ATPase Contributes to Dislocation of Endoplasmic Reticulum-associated Degradation (ERAD) Substrates

Shoshana Bar-Nun,Guy Alalouf,Efrat Rabinovich,Michael Glickman,Carni Lipson,Avigail Atir-Lande,Monika Bajorek

doi:10.1074/jbc.m705893200

Abstract

Endoplasmic reticulum (ER)-associated degradation (ERAD) eliminates aberrant proteins from the ER by dislocating them to the cytoplasm where they are tagged by ubiquitin and degraded by the proteasome. Six distinct AAA-ATPases (Rpt1-6) at the base of the 19S regulatory particle of the 26S proteasome recognize, unfold, and translocate substrates into the 20S catalytic chamber. Here we show unique contributions of individual Rpts to ERAD by employing equivalent conservative substitutions of the invariant lysine in the ATP-binding motif of each Rpt subunit. ERAD of two substrates, luminal CPY*-HA and membrane 6myc-Hmg2, is inhibited only in rpt4R and rpt2RF mutants. Conversely, in vivo degradation of a cytosolic substrate, DeltassCPY*-GFP, as well as in vitro cleavage of Suc-LLVY-AMC are hardly affected in rpt4R mutant yet are inhibited in rpt2RF mutant. Together, we find that equivalent mutations in RPT4 and RPT2 result in different phenotypes. The Rpt4 mutation is manifested in ERAD defects, whereas the Rpt2 mutation is manifested downstream, in global proteasomal activity. Accordingly, rpt4R strain is particularly sensitive to ER stress and exhibits an activated unfolded protein response, whereas rpt2RF strain is sensitive to general stress. Further characterization of Rpt4 involvement in ERAD reveals that it participates in CPY*-HA dislocation, a function previously attributed to p97/Cdc48, another AAA-ATPase essential for ERAD of CPY*-HA but dispensable for proteasomal degradation of DeltassCPY*-GFP. Pointing to Cdc48 and Rpt4 overlapping functions, excess Cdc48 partially restores impaired ERAD in rpt4R, but not in rpt2RF. We discuss models for Cdc48 and Rpt4 cooperation in ERAD.

Highlights

Fore must be eliminated [2]
In the endoplasmic reticulum (ER),4 quality control mechanisms assure that aberrant proteins are not exported; instead, they are usually dislocated to the cytosol and degraded by the ubiquitin-proteasome system in a process termed ER-associated protein degradation (ERAD; for reviews see Refs. 3–5)
The six Rpt AAA-ATPases at the base of the 19S are functionally non-equivalent, as shown by a comparative study of equivalent conservative mutations in the invariant lysine in the Walker A motif of each of the six RPT genes. These strains exhibit diverse phenotypes vis-a-vis growth sensitivity to temperature and amino acid analogs, protein degradation in vivo and proteolytic activities of purified proteasomes in vitro [31]. To assess whether their non-equivalence could be extended to ERAD, possibly uncovering unique roles for proteasomal ATPases in this pathway, we tested the degradation of ERAD substrates in wild-type, in rpt2RF, rpt3R, rpt4R, rpt5R, rpt6R and in rpt1S [31]

Summary

Introduction

Fore must be eliminated [2]. In the endoplasmic reticulum (ER),4 quality control mechanisms assure that aberrant proteins are not exported; instead, they are usually dislocated to the cytosol and degraded by the ubiquitin-proteasome system in a process termed ER-associated protein degradation (ERAD; for reviews see Refs. 3–5). The luminal ERAD substrate CPY* was stabilized in the ATPase mutants rpt4R and rpt5S but hardly in rpt2RF [10].

Results

Conclusion