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. 2003 Oct 1;31(19):5552–5559. doi: 10.1093/nar/gkg773

Figure 2.

Figure 2

Mitochondrial uptake of tRNAs in transfected cells. (A) Leishmania tropica promastigotes (4.3 × 108) were electroporated with 32P-labeled tRNATyr (500 fmol) in the absence (lanes 1–4) or presence of 10 µM CCCP (lanes 5 and 6) or 50 µM oligomycin (lanes 7 and 8). Aliquots of the transfected cells were lysed and mitochondrial fractions were treated with RNase and DNase in the absence (lanes 2, 6 and 8) or presence of 1% Triton X-100 (lane 3) or 320 µM digitonin (lane 4), before recovery and analysis of RNA in the particulate fraction. The corresponding total cellular pools (lanes 1, 5 and 7) were obtained by treating an identical aliquot of intact transfected cells with RNase before RNA isolation. (B) Transfection of promastigotes with 32P-labeled tRNAGln(CUG). Lane 1, total cellular pool; lane 2, mitochondrial fraction; lane 3, mitochondrial fraction treated with Triton X-100. (C) Kinetics of uptake of tRNATyr. Promastigotes were transfected with 500 fmol of high specific activity tRNATyr alone (lanes 1–6) or with 50 fmol of low specific activity tRNAIle (lanes 7–12). After incubation at 22°C for the indicated times, import was terminated by the addition of 10 µM CCCP, aliquots of transfected cells were lysed and RNase-resistant RNA in the mitochondrial fraction was recovered. Lane 13, input tRNATyr, 3 fmol. (D) Promastigotes were transfected with 500 fmol of high specific activity tRNAIle alone (lanes 1–6) or with 50 fmol of low specific activity tRNATyr (lanes 7–12) for the indicated times at 22°C, import terminated, and the mitochondrial uptake analyzed. Lane 13, input tRNAIle, 2 fmol. Quantitative data in (C) and (D) are graphically represented below the respective autoradiograms. Open circles, substrate alone; filled circles, substrate plus effector. (E) 32P-labeled high specific activity tRNATyr (S, 500 fmol) was co-transfected into promastigotes with low specific activity tRNAIle (E) at an E:S ratio of 0 (lanes 1 and 4), 1:10 (lanes 2 and 5) or 1:5 (lanes 3 and 6). Cells were incubated for 10 min at 22°C before analysis of total (lanes 1–3) or mitochondrial (lanes 4–6) RNA. Lane 7, input tRNATyr (1 fmol). (F) 32P-labeled high specific activity tRNAIle (S, 500 fmol) was co-transfected into promastigotes with low specific activity tRNATyr (E) at an E:S ratio of 0 (lanes 1 and 4), 1:10 (lanes 2 and 5) or 1:5 (lanes 3 and 6), and mitochondrial uptake was determined as in (E). Lane 7, input tRNAIle (1 fmol). Quantifications were performed by densitometry. The total cellular pool was taken as 100% in each case.