Figure 4.

C12orf62 is a novel COX assembly factor. C12orf62 binds to COX1 and overexpression of C12orf62-GFP and C12orf62-TAP results in reduced COX levels and activity. (a) Protein levels of subunits of five respiratory chain complexes. HEK293 cells were induced by doxycycline to overexpress C12orf62-GFP and -TAP (Materials and methods and Additional file 2). SDS-PAGE blots were immunodecorated with indicated antibodies. (b) The effect of C12orf62 overexpression on the COX holocomplex. Blue Native (BN)-PAGE analysis followed by immunodetection of complex IV (CIV) subunit COX1 and complex II subunit SDHA from C12orf62-GFP overexpressing HEK293 cells. (c) Overexpression of C12orf62-GFP affects levels of newly synthesized COX proteins. ³⁵S labeling of mitochondrial translation products from HEK293 cells overexpressing C12orf62 versus non-induced control cells. To confirm equal loading, gels were rehydrated and stained with Coomassie Brilliant Blue G-250 (CBB). Expression of the transgene and protein loading was confirmed with SDS-PAGE followed by western blotting (WB) and incubations with indicated antibodies. The loading was carried out twice (Table S9 in Additional file 2). (d) C12orf62 interacts with COX1. C12orf62-TAP was affinity purified from HEK293 cells. The purified C12orf62-TAP (eluates) were analyzed with SDS-PAGE and western blotting for co-purified proteins by probing the membranes with the indicated antibodies. Non-induced cells were used as a control. The efficiency of the pull-down was tested with the TAP-tag recognizing CBP (calmodulin binding peptide) antibody. Asterisks denote signals from previous incubations.