The endomembrane reticulum (ER) is largely reorganized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 ORF3a and membrane (M) protein expression affects ER-derived structures including cubic membrane and double membrane vesicles in coronavirus-infected cells; however, the molecular mechanisms underlying ER remodeling remain unclear. We introduced a 'plug and playable' proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged SARS-CoV-2 ORF3a and M

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... oteins. Through mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral proteins using Spot-TurboID workflow, 117 and 191 proteins were robustly determined as ORF3a and M interactomes, respectively, and many, including RNF5 (E3 ubiquitin ligase), overlap with the mitochondrial-associated membrane (MAM) proteome. RNF5 expression was correlated to ORF3a ubiquitination. MAM formation and secreted proteome profiles were largely affected by ORF3a expression. Thus, SARS-CoV-2 may utilize MAM as a viral assembly site, suggesting novel anti-viral treatment strategies for blocking viral replication in host cells.