Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation

Dara E Leto; David W Morgens; Lichao Zhang; Christopher P Walczak; Joshua E Elias; Michael C Bassik; Ron R Kopito

doi:10.1016/j.molcel.2018.11.015

Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation

Mol Cell. 2019 Jan 17;73(2):377-389.e11. doi: 10.1016/j.molcel.2018.11.015. Epub 2018 Dec 20.

Authors

Dara E Leto¹, David W Morgens², Lichao Zhang³, Christopher P Walczak¹, Joshua E Elias³, Michael C Bassik⁴, Ron R Kopito⁵

Affiliations

¹ Department of Biology, Stanford University, Stanford, CA 94305, USA.
² Department of Genetics, Stanford University, Stanford, CA 94305, USA.
³ Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.
⁴ Department of Genetics, Stanford University, Stanford, CA 94305, USA; Program in Chemistry, Engineering and Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA 94305, USA.
⁵ Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address: [email protected].

Abstract

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Protein 9 / genetics*
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats*
Endoplasmic Reticulum-Associated Degradation* / drug effects
Endoplasmic Reticulum-Associated Degradation* / genetics
Genome-Wide Association Study / methods*
HEK293 Cells
Humans
K562 Cells
Kinetics
Proteasome Endopeptidase Complex / genetics
Proteasome Endopeptidase Complex / metabolism*
Protein Folding
Proteolysis
Proteostasis* / drug effects
Proteostasis* / genetics
Ricin / pharmacology
Substrate Specificity
Ubiquitin-Protein Ligases / genetics
Ubiquitin-Protein Ligases / metabolism
Ubiquitination

Substances

Ricin
SYVN1 protein, human
Ubiquitin-Protein Ligases
CRISPR-Associated Protein 9
Proteasome Endopeptidase Complex

Abstract

Publication types

MeSH terms

Substances

Grants and funding