FIGURE 9:

Reduced energized ANT transport in the absence of OXPHOS assembly and function. (A) Mitochondria (50 μg) from wt cells treated with 15 μg/ml Dox for 7 d or from cells chased with fresh medium for 3 or 20 d were immunoblotted as listed. (B) Densitometry analyses of protein steady-state levels from A. Expression relative to untreated wt (galactose) ± SEM (n ≥ 6). (C) NADH dehydrogenase (activity/μg/min ± SEM; n ≥ 4), (D) cytochrome c reductase (μmol cytochrome c reduced/min/mg ± SEM; n = 4), and (E) cytochrome c oxidase (activity/μg/min ± SEM; n ≥ 4) activity of DDM-solubilized mitochondrial extracts. (F) Mitochondria (200 μg) from wt cells treated with 15 μg/ml Dox for 7 d or from cells chased with fresh medium for 3 or 20 d were solubilized with 1.25% (wt/vol) digitonin, resolved by 2D BN/SDS–PAGE, and immunoblotted for ANTs and the indicated respiratory complexes. (G) Mitochondria (100 μg) from wt cells untreated and grown in glucose (Glu), treated with 15 μg/ml Dox for 7 d or from cells chased with fresh medium for 3 or 20 d were fused with liposomes internally preloaded with 5 mM of unlabeled ADP. Uptake of [¹⁴C]ADP (nM/mg protein/min ± SEM; n = 3). (H) The uptake of [¹⁴C]ADP (nM/mg protein/min ± SEM; n = 6) into energized mitochondria (500 μg) was determined in the presence of 5 mM glutamate and malate.