Figure 8
High molecular weight complexes containing MCAD are detectable by BN-PAGE. (A) MCAD was radiolabeled by in vitro transcription/translation, followed by incubation for 5, 10, 30 and 60 min with isolated 143B control mitochondria. Mitochondria were solubilised in 1% (v/v) TX-100 or 1% (w/v) digitonin, followed by BN-PAGE analysis. MCAD can be detected in complexes of ~175 kDa (MCAD homotetramer) and ~450 kDa (mitochondrial Hsp60 complex), as well as unknown complexes of ~500 kDa (marked *) and ~1,500 kDa (marked #). The intensities of both the ~500 kDa (*) and ~1,500 kDa (#) MCAD-containing complexes (relative to the ~450 kDa mitochondrial Hsp60 complex) was reduced over the 60 min import time course (p = 0.016 and 0.005 respectively). (B) SDS-PAGE showing MCAD in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. MCAD is imported in a mitochondrial membrane potential (Δψm) dependent manner, with the levels of mature MCAD protein (m) similar at each time point for both the TX-100 and DIG experiments. (C) BN-PAGE and Western blot analysis of HepG2 cell mitochondria solubilised in 1% (w/v) digitonin (DIG), 0.2% (w/v) dodecyl maltoside (DDM) or 1% (v/v) TX-100. Anti-NDUFA9 antibodies detect monomeric complex I and the CI/CIII2/CIVn and CI/CIII2 OXPHOS supercomplexes, while anti-MCAD antibodies detect MCAD in complexes of ~130 kDa, ~175 kDa (homotetramer), ~450 kDa (mitochondrial Hsp60 complex) and ~1,000 kDa (*).
