Figure 6.
Import of human tRNALys(UUU) into Leishmania mitochondria. (A) Intra-mitochondrial distribution assay. 32P-labeled tRNALys (100 fmol) was incubated with intact mitochondria (100 µg of protein), and its sub-mitochondrial distribution was analyzed. (B) Promastigotes were transfected with 500 fmol of high specific activity tRNALys in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 50 µM oligomycin. Total and mitochondrial pools were recovered as in Figure 2. (C) Effect of tRNATyr and tRNAIle on import (top) or binding (bottom) of tRNALys into or on mitoplasts in vitro. Lane 1, tRNALys substrate alone; lane 2, substrate plus tRNATyr effector; lane 3, substrate plus tRNAIle effector. (D) Effect of tRNATyr and tRNAIle on internalization of tRNALys into mitochondria of transfected cells. Co-transfection of promastigotes with high specific activity human tRNALys and low specific activity tRNAIle (top) or tRNATyr (bottom) was performed at effector:substrate ratios of 0 (lanes 1 and 4), 1:10 (lanes 2 and 5) and 1:5 (lanes 3 and 6), respectively. The total cellular pool (lanes 1–3) and mitochondrial pool (lanes 3–6) were recovered in each case. (E) Effect of tRNALys on import (top) or binding (bottom) of tRNATyr into or on mitoplasts. Import reactions contained 50 fmol of substrate and mitoplasts from 100 µg of mitochondria; corresponding values for binding assays were 10 fmol and 50 µg, respectively. Substrate:effector ratio was 10:1. Lane 1, tRNATyr alone; lane 2, tRNATyr plus tRNAIle effector; lane 3, tRNATyr plus tRNALys effector; lane 4, input tRNATyr, 1.0 and 0.5 fmol, respectively. (F) Effect of tRNALys on import (top) or binding (bottom) of tRNAIle. Assays were performed as in (E), except that tRNAIle was used as substrate. Lane 1, substrate alone; lane 2, substrate plus tRNATyr effector; lane 3, substrate plus tRNALys effector; lane 4, input tRNAIle, 5 fmol.