(B) data are representative of two independent experiments. (B-C) Plots of INSIG1-GFP turnover assessed by protein synthesis shut-off and flow cytometry analysis of cells expressing the indicated single or double sgRNAs. (A) Plots of INSIG1-GFP turnover assessed by protein synthesis shut-off and flow cytometry analysis of cells expressing the indicated sgRNAs data are the mean В± SEM of three independent experiments. Arrows indicate vOTU-resistant Ub n -GFP u* representative of two independent experiments. (I) Ub n -GFP u* conjugates affinity captured with immobilized Halo-TRABID NZF1 and treated with the indicated DUBs for 1 hr. (F-G) Ub n-GFP u* conjugates affinity captured from lysates treated with UPS inhibitors (F-G) and expressing exogenous UBE3C variants (G) representative of three (F) or two (G) independent experiments. Top: Immunoblots bottom: GFP u* quantification representative of two independent experiments. (E) GFP u* turnover in UBE3C KO cells expressing indicated UBE3C variants. (D) Plots of GFP u* turnover assessed at the indicated times after protein synthesis shut-off by flow cytometry analysis of cells expressing the indicated single or double sgRNAs. Dashed line indicates removal of irrelevant gel lanes. (C) Immunoblots of cytosolic (cyto) and membrane (memb) cell fractions representative of three independent experiments. Treatments were 3 hr representative of two independent experiments. (B) Immunoblots of TX-100 soluble (S) and insoluble (I) cell fractions. (A) Plots of GFP u* turnover assessed by protein synthesis shut-off and immunoblotting of lysates from cell expressing the indicated sgRNAs data are the mean of three independent experiments В± SEM. Cytosolic E3 ligases identified in the genetic screens are in the dashed box. Analysis includes genes in the indicated categories at в‰¤1% FDR in at least one screen. Gene score signs were assigned based on guide enrichment, with a negative sign indicating guide disenrichment in the GFP high population relative to the GFP low population and a positive sign indicating guide enrichment in the GFP high population relative to the GFP low population. (F) Hierarchical clustering of genes by gene score. Each data point represents the disruption of a specific gene for a given substrate indicated by color. Linear least-squares regression of gene effects from all four screens plotted against normalized degradation rates, calculated as the slope of the log-transformed decay kinetics, obtained by translation shut-off analysis in cells expressing single sgRNAs. (E) Screen metrics are well correlated with substrate degradation rates. (C) Cells were treated with inhibitors for 3 hr and median fluorescence intensity (MFI) was measured by flow cytometry analysis at the indicated times after inhibiting protein synthesis with emetine representative of two independent experiments.

(B) Fluorescence histograms of the indicated cell lines treated +/в€’ doxycycline (dox) for 16 hr before adding inhibitors for 3 hr representative of three independent experiments. Triangles represent N-glycosylation sequons -SH represents cysteines. (A) Schematic of the reporters used in this study. 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.Д¬opyright В© 2018 Elsevier Inc. 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). 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.

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