Figure 6. Ovarian carcinoma immunoreactive antigen domain-containing protein 1 (OCIAD1) regulates IMMP2L-dependent proteolytic processing of cytochrome c₁ (CYC1).

(A) 2D-native/SDS-PAGE analysis of mitochondrial membranes isolated from K562 control and OCIAD1 knockdown cells and immunoblotted with CYC1 and PHB2 antibodies. CIII₂ assemblies from OCIAD1 knockdown cells contained immature CYC1 of higher molecular weight. PHB2 staining served as an internal molecular size reference. Light blue horizontal lines represent the size of putative precursor (p), intermediate (i), and mature (m) CYC1. White vertical lines represent the different high-order CIII₂ assemblies. (B) Extracted MS2 fragment ion chromatograms (XIC) for three diagnostic CYC1 peptides detected by diaPASEF mass spectrometry in blue-native polyacrylamide gel electrophoresis (BN-PAGE) gel slices excised from control cells, OCIAD1 knockdown cells, and OCIAD1 knockdown cells rescued with wildtype OCIAD1. Individual peptides displayed highly correlated fragment ion co-elution profiles strongly supportive of peptide identification. The TPQAVALSSK⁺⁺ peptide (bottom panel), located at the N-terminus of the CYC1 hydrophobic sorting sequence, was only identified in CIII₂ assemblies from OCIAD1 knockdown cells. Conversely, the SDLELHPPSYPWSHR⁺⁺⁺ peptide (middle panel), which uniquely identifies the N-terminus of mature CYC1 but is not present in the tryptic digest of the CYC1 precursor, was reliably detected in CIII₂ assemblies from control and OCIAD1 knockdown cells rescued with wildtype OCIAD1, but not from OCIAD1 knockdown cells. An internal peptide (LFDYFPKPYPNSEAAR⁺⁺⁺, top panel) common to all CYC1 species (precursor, intermediate, mature) was detected in all cell lines, albeit at lower levels in OCIAD1 cells as expected.