1. MARCH6 and TRC8 are required for the degradation of selected ER proteins following SPP‐mediated intramembrane proteolysis
MARCH6 and TRC8 are required for the degradation of selected ER proteins following SPP‐mediated intramembrane proteolysis Volcano plot showing TMT mass spectrometry analysis of whole‐cell proteome of mCherry‐CL1 cells compared to combined MARCH6/TRC8 null cells. Significance is shown in the y‐axis (log(2) q‐value) and log(2)‐fold change on the x‐axis. Proteins that were highly enriched in the combined MARCH6/TRC8 null cells and not in the single MARCH6 or TRC8 KO clones (detailed in Dataset EV2) are shown in purple.Schematic of membrane topology of the tail‐anchored protein, HO‐1, that fails to insert correctly, or before and after intramembrane cleavage by SPP, in comparison with the amphipathic mCherry‐CL1 degron.[35S]methionine/cysteine‐radiolabelling of HO‐1 in HeLa mCherry‐CL1 cells (WT) or the combined MARCH6/TRC8 null cells. Cells were pulse‐radiolabelled for 10 min and immunoprecipitated for HO‐1 from detergent lysates at the times indicated. Cells were treated with or without the SPP inhibitor (SPP inb), 10 μM (Z‐LL)2 ketone for 24 h. Full‐length (black) and SPP‐cleaved form of HO‐1 (blue) are indicated. The asterisk represents non‐specific band observed in the HeLa mCherry‐CL1 cells (see also Appendix Fig S5).[35S]methionine/cysteine‐radiolabelling of HO‐1 in wild‐type HeLa cells, in HeLa TRC8 KO cells (see Appendix Fig S5C and D) or in the TRC8 KO clone depleted for MARCH6 by sgRNA (M6 KO), as described. Full‐length and SPP‐cleaved form of HO‐1 are indicated.[35S]methionine/cysteine‐radiolabelling of HO‐1 in HeLa cells (WT) or combined MARCH6/TRC8 null cells, with or without overexpressed SPP. The SPP‐cleaved form of HO‐1 is indicated.Schematic for mechanism of HO‐1 quality control at the ER membrane, requiring both MARCH6 and TRC8 to ubiquitinate misinserted or cleaved HO‐1, targeting HO‐1 for proteasome‐mediated degradation.
Sandra Stefanovic‐Barrett et al. EMBO Rep. 2018;embr
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