Fig 1: Levels of Endogenous CYP51A1 and TMUB2 Are Regulated by the RNF185-MBRL Complex(A and B) Protein extracts from RNF185, MBRL, and HRD1 KO (A) HEK293 and (B) HeLa cells expressing either an EV or cDNAs encoding the WT or CI version of the indicated protein were analyzed by SDS-PAGE and immunoblotting. Extracts from parental cells left untreated or treated with the p97 inhibitor CB-5083 (CB) (4 h; 2.5 µM) were also analyzed. Relative CYP51A1 and TMUB2 levels are displayed below the respective blot, normalized to the GAPDH loading control.(C) Endogenous CYP51A1 co-precipitates specifically with RNF185/MBRL complex. The indicated FLAG-tagged proteins (x axis) were precipitated from untreated or CB-5083-treated cells, and eluted proteins were analyzed by mass spectrometry. The y axis shows the log2 fold enrichment of endogenous CYP51A1 in the FLAG precipitates versus untagged control.See also Figure S5.
Fig 2: RNF185-MBRL and TEB4 Recognize Distinct Features on Their Membrane Substrates(A) Schematic overview of the CYP51A1TM chimeras. The AH and the TMD from the Erg11TM ERAD substrate are indicated in purple.(B) The levels of CYP51A1TM chimeras were analyzed by flow cytometry (based on GFP fluorescence) in the absence or presence of the p97 inhibitor CB-5083 (4 h; 2.5 µM).(C) The levels of CYP51A1TM and the chimera CYP51A1TMErg11TMD were analyzed by flow cytometry (based on GFP fluorescence) in cells depleted for the indicated genes.(D) Degradation of CYP51A1TMErg11TMD analyzed upon inhibition of protein synthesis with CHX in cells depleted for the indicated genes. Cell extracts were analyzed by SDS-PAGE and immunoblotting. The graph shows the average of three experiments; error bars represent the standard deviation.See also Figure S6.
Fig 3: CYP51A1TM Ubiquitination and Degradation Are Dependent on RNF185 and Membralin(A) RNF185 and Membralin are essential for CYP51A1TM degradation. Degradation of CYP51A1TM was analyzed upon inhibition of protein synthesis with cycloheximide (CHX) in parental, RNF185, MBRL, and HRD1 KO cells. Cell extracts were analyzed by SDS-PAGE and immunoblotting. The graph shows the average of three experiments; error bars represent the standard deviation.(B) UBE3C promotes efficient CYP51A1TM degradation. Degradation of CYP51A1TM in cells lacking the indicated genes was analyzed as in (A).(C) The E2s UBE2K and UBE2D3 are involved in CYP51A1TM degradation. Degradation of CYP51A1TM in cells depleted for the indicated genes was analyzed as in (A).(D) Ubiquitination of CYP51A1TM is dependent on RNF185 and MBRL. CYP51A1TM was immunoprecipitated from parental cells or cells lacking the indicated genes and analyzed by SDS-PAGE followed by immunoblotting with anti-HA and anti-ubiquitin antibodies. Parental cells lacking CYP51A1TM substrate were used as a negative control.(E) UBE2K facilitates polyubiquitination of CYP51A1TM. CYP51A1TM ubiquitination in cells depleted for the indicated E2 enzymes was analyzed as in (D).See also Figure S3.
Fig 4: RNF185 and Membralin Form a Novel ERAD Complex(A) Proteins co-precipitating with MBRL-3xFLAG (x axis) and 3xFLAG-RNF185 (y axis) as analyzed by mass spectrometry. The enrichment of proteins associated to FLAG-tagged baits over an untagged control was used to calculate the log2 fold changes. Proteins enriched above an arbitrary cutoff of 4 in both MBRL and RNF185 IPs are annotated (in orange).(B) Proteins co-precipitating with endogenous Membralin-mNG as detected by mass spectrometry. The x axis shows the log2 fold change of MBRL-mNG versus untagged control cell line; the y axis shows the -log10 p value estimated by the SignificanceB analysis (Cox and Mann, 2008). Cutoff was arbitrarily set to a log2 (fold change) of 4 with a -log10 (p) of 5. Significantly enriched proteins are annotated (in orange).(C) Western blotting validation of the interactions identified in (B) by mNG-Trap pull-down (PD) of endogenously tagged Membralin-mNG. Endogenously tagged HRD1-mNG was used as specificity control.(D) Analysis of CYP51A1TM levels in cells depleted for TMUB1, TMUB2, or both. CYP51A1TM was analyzed by flow cytometry (based on GFP fluorescence).(E) Size exclusion chromatography of detergent-solubilized membranes of HeLa cells containing endogenously tagged MBRL-mNG (MBRL-mNG). Crude membranes were solubilized in 1% DDM + 0.1% CHS, and solubilized material was applied to a Superose 6 10/300 GL column. Elution fractions were analyzed by SDS-PAGE and western blotting with the indicated antibodies.(F) Fraction 9 from (E) was subjected to immunoprecipitation using mNG-Trap beads. Eluted material was analyzed by western blotting for the proteins indicated.(G) Membralin mediates the interaction between RNF185 and TMUBs. Immunoprecipitation of 3xFLAG-RNF185 in cells with the indicated gene deletions is shown. Eluted proteins were analyzed by SDS-PAGE and immunoblotting for the proteins indicated. The asterisk (*) indicates a truncated MBRL product.See also Figure S4.
Fig 5: A Genome-wide CRISPR-Cas9 Screen Identifies Components Required for CYP51A1TM Degradation(A) Workflow of the CRISPR-Cas9 genome-wide screen.(B) Significance score of the genes analyzed in the screen calculated by the MAGeCK algorithm. The x axis represents the genes in alphabetical order. The y axis shows the -log(aRRA) significance value. The -log(aRRA) cutoff was arbitrarily set at 9 (dashed line). Significantly enriched genes are annotated.(C) Overview of the enriched genes identified and their proposed function.(D) Validation of several screen hits using independent sgRNAs. Levels of CYP51A1TM were analyzed by flow cytometry (based on GFP fluorescence).(E) Degradation of CYP51A1TM depends on RNF185, MBRL, SPP, and UBE2K. CYP51A1TM levels were assessed by flow cytometry (based on GFP fluorescence) in parental control cells and clonal RNF185, MBRL, SPP, UBE2K, and HRD1 KO cells expressing cDNAs encoding either WT, a CI mutant, or an EV.See also Figure S2.
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