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

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 foldedmembrane 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-reticulumassociated 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.