Fig 1: EMC-Dependent Proteins Contain Various Numbers of TMDs(A) All 971 membrane proteins with UniProt “Transmembrane” feature key were categorized based on the number of their TMDs. The x axis shows the log2 ratios of protein levels in EMC6-KO over WT cells, and the y axis shows the number of TMDs. EMC-dependent proteins (red dots) and EMC members (blue dots) are highlighted. The proteins with more than 11 TMDs were clustered as one group (=12).(B) The number of identified EMC-dependent proteins (red bars) and EMC-independent proteins (blue bars) over the total number of transmembrane proteins with the same number of TMDs within 971 transmembrane proteins. The inside panel shows the relative frequency for three groups: single TMD, 2–4 TMDs, and more than 4 TMDs among identified 36 EMC-dependent, 171 EMC-independent, and all 971 transmembrane proteins, respectively.(C) Validation of EMC-dependent proteins. Expression levels of FDFT1, CD9, and ATP6V0A1 in WT, EMC4-Mut, and EMC6-KO cells were analyzed by immunoblot using antibodies targeting endogenous proteins. ATP6V0A1, ZFPL1, FZD6, and SLC43A3 with the indicated fusion tags were expressed in WT cell lines and EMC-deficient cell lines via transient transfection. Actin served as a loading control. FLAG-N, N-terminal triple-FLAG tag; FLAG-C, C-terminal triple-FLAG tag; 1D4-C, C-terminal 1D4 tag.(D) Validation of EMC-independent proteins. Endogenous SEC61A1 in WT cell lines and EMC-deficient cell lines were detected via immunoblot using an anti-SEC61A1 antibody, while other indicated EMC-independent proteins were expressed in WT and EMC-deficient cell lines via transient transfection and detected via fused FLAG or HA tags by immunoblot analysis. HA-C, C-terminal triple-HA tag.In (C) and (D), representative images were from one of three independent experiments.