Figure 3. Mitochondrial inner membrane localization and topology of MCUR1 and its interaction with MCU.

Confocal images of HeLa cells transiently co-transfected for 48 hrs with (a) GFP-tagged MCUR1 and DsRed Mito plasmids or (b) mRFP-tagged MCUR1 and EYFP-Mito. Scale bar: 20 μm. (c) Immunoblot analysis of Flag-tagged MCUR1 in HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (OMM protein), COII (integral membrane marker) and Flag. (d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane (OMM) permeabilization and appearance of cytosolic cytochrome c was verified. Intact and OMM permeabilized samples were exposed to Proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1. (e) Reciprocal co-immunoprecipitation of MCU-GFP and MCUR1-Flag transiently expressed in COS7 cells. Representative of four independent experiments. (f) Co-immunoprecipitation of MICU1-Flag with MCU-GFP but not with MCUR1-GFP transiently expressed in COS7 cells. Representative of four independent experiments. (g) Immunoprecipitation of LETM1-GFP with anti-LETM1 failed to pull down MCUR1-Flag in transiently-transfected COS7 cells. (h) Immunoprecipitation with Flag antibody pulled down LETM1-Flag or MCU-Flag transiently expressed in COS7 cells (IP lanes 2 and 3, lower panel), but only co-immunoprecipitated MCUR1-V5 in the MCU-Flag expressing cells (IP lanes 4 vs 5, upper panel), despite lower expression of MCU-Flag in MCUR1-cotransfected cells (compare MCU-Flag and LETM1-Flag expression in MCUR1-cotransfected cells in lysate lanes 4 and 5, bottom panel). Representative of three independent experiments.