Figure 2.

Ablation of F1α and F1β does not affect mitochondrial transfer RNA import but impairs oxidative phosphorylation. (A) As in Fig 1, but a F1α-RNAi cell line was analysed. Left middle panel: RNAi-induced ablation of the F1α mRNA was verified by northern blot analysis. The EtBr-stained rRNA region acts as a loading control. Left bottom panel: immunoblot containing total cellular (Tot) and mitochondrial fractions (Mit) of Tet-uninduced and Tet-induced RNAi cells probed with anti-sera against F1β, eEF1a and VDAC. Cells were analysed after 46 and 92 h of Tet induction. (B) As in A but a F1β-RNAi cell line was analysed. (C) In organello, ATP production was determined in digitonin-isolated mitochondria of F1α (top panel) and F1β RNAi cell lines (lower panel). Uninduced cells (−Tet) are shown on the left, and induced cells (+Tet) are shown on the right. The substrate tested is indicated at the top, and the additions of antimycin and atractyloside are shown at the bottom. ATP production in mitochondria isolated from uninduced cells tested without antimycin or atractyloside was set to 100%. The bars represent means expressed as percentages from at least three independent inductions. Standard errors are indicated. Cyto, cytosolic; eEF1α, elongation factor 1α; EtBr; ethidium bromide; IPTG, isopropyl-β-D-thiogalactopyranoside; mRNA, messenger RNA; RNAi, RNA interference; rRNA, ribosomal RNA; Tet, tetracycline; tRNA, transfer RNA; VDAC, voltage-dependent anion channel.