Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function
Brian G. Peterson,
Jiwon Hwang,
Jennifer E. Russ,
Jeremy W. Schroeder,
P. Lydia Freddolino,
Ryan D. Baldridge
Affiliations
Brian G. Peterson
Department of Biological Chemistry, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA
Jiwon Hwang
Department of Biological Chemistry, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA
Jennifer E. Russ
Department of Biological Chemistry, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA
Jeremy W. Schroeder
Department of Biological Chemistry, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA
P. Lydia Freddolino
Department of Biological Chemistry, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA; Cellular and Molecular Biology Program, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA; Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
Ryan D. Baldridge
Department of Biological Chemistry, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA; Cellular and Molecular Biology Program, University of Michigan Medical School, 1150 W Medical Center Drive, Ann Arbor, MI 48109, USA; Corresponding author
Summary: Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-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 identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane.
