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Fig. 5. LETM1 is required for the assembly of the respiratory chain. (A) HeLa cells transfected with siRNA targeting GFP (control) or LETM1 were incubated for 60 minutes with Mitotracker Red CMXRos at the indicated concentrations, washed, fixed and analyzed by immunofluorescence microscopy with anti-mitofilin antibody. The uptake of Mitotracker Red CMXRos clearly decreased in the LETM1 knockdown cells. Fluorescent images were obtained by confocal microscopy. (B) Total cell lysates from cells transfected with siRNA targeting GFP (control) or LETM1 were subjected to SDS-PAGE and immunoblotted using antibodies to the indicated proteins. LETM1 knockdown caused reduction in the supercomplexes of the respiratory chains. (C) Mitochondrial fractions from cells transfected with siRNA targeting GFP (control) or LETM1 were digitonin-solubilized, centrifuged, subjected to Blue-Native gel electrophoresis and immunoblotted with antibodies against the indicated proteins. Arrowhead indicates the supercomplexes of complexes I, III and IV. Asterisk indicates a nonspecific band that was found even when BCS1L was repressed (see Fig. 8B). (D) Mitochondrial fractions from cells transfected with siRNA targeting GFP (control) or LETM1 were treated with alkaline buffers ranging in their pH values between 11.5 and 12.5, centrifuged for separation into supernatant (S) and pellet fractions (P), and immunoblotted with antibodies against the indicated proteins. The membrane association of core1 protein was affected by LETM1 downregulation.
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