Stepwise transfer of tRNA through the double membrane of Leishmania mitochondria

Import of tRNA into Leishmania mitochondria involves transfer through a dou ble membrane barrier. To examine whether specific sorting mechanisms for in dividual tRNAs direct them to different mitochondrial compartments, the dis tribution of tRNA transcripts, internalized in vitro, was examined by subor ganellar fractionation. Significant amounts of tRNA(Tyr) were localized in the matrix and on the outer face of the inner mitochondrial membrane. With time, the matrix:membrane ratio increased. Translocation through the inner membrane apparently required the presence of a specific signal in the D arm of tRNA(Tyr), and tRNA(Gln)(CUG), lacking this sequence, was excluded. Hyd rolysis of ATP was necessary at both the outer and inner membranes. However , the protonophores carbonylcyanide m-chlorophenylhydrazone and nigericin, the K+ ionophore valinomycin, and the F1F0 ATPase inhibitor oligomycin had only marginal effects on uptake through the outer membrane but severely inh ibited inner membrane translocation, indicating the unusual requirement of both the electrical and chemical components of the electromotive force gene rated across the inner membrane, The results are consistent with a mechanis m involving stepwise transfer of tRNA through distinct outer and inner memb rane channels.