Figure 7
The assembly of the nuclear-encoded subunit COX VIa-L into mature complex IV and the OXPHOS supercomplexes is reduced in 143B MCAD knockout mitochondria. COX VIa-L was radiolabeled by in vitro transcription/translation, followed by incubation for 10 and 60 min with isolated 143B control (CON) or 143B MCAD knockout (KO) mitochondria. (A) SDS-PAGE showing COX VIa-L in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. COX VIa-L is imported efficiently into both CON and MCAD KO mitochondria. (B) BN-PAGE showing the assembly of COX VIa-L into the late-stage intermediate (CIVLSI) and mature complex IV (CIVm) [following solubilisation in 1% (v/v) TX-100, left] or the CI/CIII2/CIVn supercomplexes [following solubilisation in 1% (w/v) digitonin, right]. The amount of newly-translated COX VIa-L assembled into CIVm and the CI/CIII2/CIVn supercomplexes was significantly less in MCAD KO mitochondria compared to CON mitochondria after import times of both 10 and 60 min. (C) Quantitation of assembled COX VIa-L into CIVm (n = 4) and the CI/CIII2/CIVn supercomplexes (n = 3). Data is mean ± s.d. **p < 0.01.
