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. 2006 Nov 14;103(48):18362–18367. doi: 10.1073/pnas.0606449103

Fig. 2.

Fig. 2.

Implication of VDAC in mitochondrial tRNA import. (A and C) In vitro import of tRNA into isolated mitochondria. Labeled in vitro-transcribed tRNAAla (105 cpm) was incubated with isolated mitochondria in the absence (−) or presence (+) of 5 μl of antibodies against VDAC or LeuRS (A) and 5 or 10 μM RuR or 10 or 100 μM ATR (C). Incubations performed in the absence (−) or presence (+) of 5 mM ATP were used as control experiments. In, input RNAs (100 cpm). The corresponding ethidium bromide-stained gels are shown below the autoradiograms. Incubation of tRNAAla transcript with mitochondria in the presence of increasing concentration of VDAC antibodies shows that 1 μl of VDAC antibodies completely inhibits import (data not shown). (B and D) In vitro import of protein into isolated mitochondria. Labeled in vitro-synthesized GluRS targeting sequence fused to GFP was incubated with mitochondria in the absence (−) or presence (+) of 5 μl of antibodies against TOM20, VDAC, or LeuRS (B) or 10 μM RuR or 100 μM ATR (D). Upon import into mitochondria, the targeting presequence is cleaved off from the precursor protein (p), and a smaller protein corresponding to mature form (m) appears. As a control, protein import was shown to depend on mitochondrial membrane potential, as demonstrated by inhibition of import by valinomycin. In, input radiolabeled protein GluRS-GFP. (E) Binding of tRNAAla to isolated mitochondria depends on trypsin-sensitive proteins. To remove proteins exposed on the outer membrane, isolated mitochondria were treated with increasing concentrations of trypsin (0, 2, 4, and 8 μg of trypsin per mg of mitochondrial proteins) as described (14). Binding was performed under the same conditions as for tRNA import except that the RNase digestion step was omitted after incubation with labeled tRNAAla. As previously reported, the amount of bound tRNAs onto mitochondria is ≈2–5% of the input (compared with 0.2–0.5% of the input for RNase-protected imported tRNAs). Corresponding mitochondrial protein samples were analyzed by Western blot, using antibodies directed against VDAC, TOM20, and TOM40. For graphical representation of the results, the amounts of bound tRNAs (●), TOM20 (▴), TOM40 (■), and VDAC (○) were quantified with MacBAS 100 software.