Research Paper Volume 16, Issue 6 pp 5501—5525

Figure 4. In vivo association between EMC and VDAC1. (A) ER membrane proteins PIGK, PIGU, OST48 and RPN2 were tagged on their cytoplasmic side. Mitochondrial outer membrane protein SAM50 was tagged at its C-terminus. Luminescence intensities were measured to quantify the strength of the protein-protein interactions, and the intensity of VDAC1 with EMC7 was set as 100 for each experiment. The result represents mean ± s.e.m from n = 3 independent experiments. (B) A silver staining image of the SDS-PAGE gel for the human EMC complex purified from Expi293 cells that lack endogenous EMC7 but express C-terminal Flag-tag EMC7^1–156 ectopically. (C) Representative reference-free 2D negative staining-EM average of the purified mutant EMC. (D) The truncation mutant EMC7^1–156 attenuates the formation of EMC-VDAC1 complex. Left: Silver-stained SDS-PAGE of the purified EMC complex. Right: Western blot analysis of the purified EMC complex and whole cell lysate using antibodies as indicated. (E) BiFC assay examining the location of the EMC-VDAC1 complex. Proteins are fused either to the N-terminal fragment (-YFPVN) or to the C-terminal fragment (-YFPVC) of YFP. Mitochondria were stained by MitoTracker probes. BF: Bright field. Scale bar: 5 μm. (F) Purification of the EMC complex in the native lipid environment. Left: Silver-stained SDS-PAGE of the purified EMC complex in SMALPs with or without EGS crosslinker. Right: Western blot analysis of the purified EMC complex using antibodies as indicated. ^* indicates the cross-linked form of the target proteins.