RT Journal Article
SR Electronic
T1 Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.04.03.535444
DO 10.1101/2023.04.03.535444
A1 Brian G. Peterson
A1 Jiwon Hwang
A1 Jennifer E. Russ
A1 Jeremy Schroeder
A1 Peter L. Freddolino
A1 Ryan D. Baldridge
YR 2023
UL http://biorxiv.org/content/early/2023/04/03/2023.04.03.535444.abstract
AB Misfolded endoplasmic reticulum proteins are degraded through a process called endoplasmic reticulum associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identified several regions required for different Hrd1 functions. Most surprisingly, we found two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we defined roles for disordered regions between structural elements that were required for Hrd1’s ability to autoubiquitinate and interact with substrate. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the endoplasmic reticulum membrane.Competing Interest StatementThe authors have declared no competing interest.