Fig. 4.

F₁β presequence competes for protein import but not for tRNA import, and unlabeled tRNA competes for tRNA import but not for protein import. (A) Labeled in vitro-synthesized GluRS-GFP protein was incubated with isolated mitochondria in the absence (−) or presence of 3 or 5 μM F₁β presequence (pF1b). After incubation, samples were treated (+) or not (−) with proteinase K (Prot. K). As a control, protein import was inhibited in the presence (+) of valinomycin (Valino). In, input-labeled protein GluRS-GFP. (B) Labeled in vitro-transcribed tRNA^Ala (10⁵ cpm) was incubated with isolated mitochondria in the absence (−) or presence of 3 or 5 μM F₁β presequence. As a control, incubations were performed in the absence (−) or presence (+) of 5 mM ATP. The corresponding ethidium bromide-stained gel is shown below the autoradiogram. In, input RNAs (100 cpm). (C) Competition experiments of tRNA^Ala import into isolated mitochondria using unlabeled tRNA^Ala as competitor (^utRNA). The experiments were performed in the absence (−) or presence (+) of 5 mM ATP. In, input RNAs (1,000 cpm). (D) Competition experiments of GluRS-GFP protein import into isolated mitochondria using unlabeled tRNA^Ala as competitor (^utRNA). As a control, protein import was run in the absence (−) or presence (+) of valinomycin (Valino). In, input-labeled protein GluRS-GFP.