Fig 5. Mic23/ApoO is co-precipitated with Mic60/Mitofilin and its import is affected by depletion of Mic60/Mitofilin and Sam50.

(A) Knockdown of Mic60/Mitofilin, Sam50 and Mic27/ApoOL was induced for 7 days using doxycycline (Dox) in the respective knockdown cell line (mflkd-2 for Mic60/Mitofilin, sam50kd-2 for Sam50 and apoolkd-2 for Mic27/ApoOL). Mitochondria were isolated and 50 μg of mitochondria per lane was incubated with ³⁵S-labeled Mic23/ApoO for indicated times. Lower panels represent western blot controls of the knockdown and loading, using antibodies against Mic60/Mitofilin, Sam50 or Mic27/ApoOL and Hsp60 or SDHA. Hsp60—heat shock protein 60, SDHA—succinate dehydrogenase, a component of the mitochondrial respiratory complex II. (B) Pulse-chase experiment was performed with 50 μg of HeLa mitochondria per lane, which were incubated with ³⁵S-labeled Mic23/ApoO for 20 min to allow formation of the Mic23/ApoO import complex. Mitochondria were reisolated, incubated in the import buffer for indicated times and analyzed by BN-PAGE and autoradiography. (C) Inducible knockdown cell line carrying shRNA that downregulates Sam50 (sam50kd-2) was induced for 7 days with doxycycline (Dox) and mitochondria were isolated from non-induced (-Dox) and induced cells (+Dox). 50 μg of mitochondrial protein per lane was solubilized in 1% digitonin buffer and analyzed by blue native (BN)-PAGE and western blot, using antibodies directed against Sam50, Mic60/Mitofilin or Mic19/CHCHD3, and, as control, SDHA (lower panels). SDHA—succinate dehydrogenase, a component of the mitochondrial respiratory complex II. Asterisks indicate three protein complexes observed after ³⁵S-labeled Mic23/ApoO import or western blot analysis of the steady state levels of Sam50, Mic60/Mitofilin and Mic19/CHCHD3. (D) Co-immunoprecipitation was performed by targeting Mic60/Mitofilin with specific antibodies and analyzing the precipitates by SDS-PAGE and western blot, using Mic60/Mitofilin, Mic23/ApoO and Tim44 antibodies. PBS was used as a negative control. Tim44—translocase of the inner mitochondrial membrane 44.