Mutations in a tRNA import signal define distinct receptors at the two membranes of Leishmania mitochondria

Nucleus-encoded tRNAs are selectively imported into the mitochondrion of Le ishmania, a kinetoplastid protozoan. An oligoribonucleotide constituting th e D stem-loop import signal of tRNA(Tyr)(GUA) was efficiently transported i nto the mitochondrial matrix in organello as well as in vivo. Transfer thro ugh the inner membrane could be uncoupled from that through the outer membr ane and was resistant to antibody against the outer membrane receptor TAB. A number of mutations in the import signal had differential effects on oute r and inner membrane transfer. Some mutants which efficiently traversed the outer membrane mere unable to enter the matrix. Conversely, restoration of the loop-closing GC pair in reverse resulted in reversion of transfer thro ugh the inner, but not the outer, membrane, and binding of the RNA to the i nner membrane was restored. These experiments indicate the presence at the two membranes of receptors with distinct specificities which mediate stepwi se transfer into the mitochondrial matrix. The combination of oligonucleoti de mutagenesis and biochemical fractionation may provide a general tool for the identification of tRNA transport factors.