Fig. 2.

TMEM177 associates with COX20 in the inner mitochondrial membrane (A) A Schematic representation of the primary sequence of TMEM177. The predicted transmembrane spans (TM) are depicted (numbers indicate amino acids residues). (B) TMEM177 localizes to mitochondria. HEK293T cells were fractionated by differential centrifugation. Different fractions were analyzed by SDS-PAGE and Western blotting. Total, total cell extract; Mito., mitochondria; Cyto., cytoplasm; ER, endoplasmatic reticulum. (C) Immunofluorescence microscopy of HeLa cells expressing TMEM177^FLAG. Cells were immunolabeled using anti-FLAG antiserum (green) and stained with MitoTracker (red) and DAPI (blue). Scale bar, 10 μm. (D) TMEM177 is an integral membrane protein. Isolated mitochondria from wild-type cells were subjected to carbonate extraction at the indicated pH, or solubilized with Triton X-100. Upon differential centrifugation, different fractions total (T), pellet (P), and supernatant (S) were analyzed by SDS-PAGE and Western blotting. (E) The C-terminus of TMEM177 is exposed to the IMS. Submitochondrial localization analysis using wild-type mitochondria or mitoplast. Sonic., sonication; Mito., mitochondria; MP, mitoplast; PK, proteinase K. (F) COX20 associates with TMEM177. Protein complexes were isolated from mitochondria from wild-type and COX20^FLAG-expressing cells by FLAG-immunoprecipitation and analyzed by SDS-PAGE and Western blotting. COX20^FLAG signals were detected using anti-FLAG antibody. Total, 1%; Eluate, 100%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)