Mutations in a tRNA Import Signal Define Distinct Receptors at the Two Membranes of Leishmania Mitochondria

Abstract

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

ACKNOWLEDGMENTS

We are indebted to Subhagata Ghosh for technical assistance, Chanchal Dasgupta for nucleotides, and Tapas Ghosh of the Bioinformatics Centre, Bose Institute, for running the FOLDRNA program.

This work was supported by a grant from the Department of Science and Technology, Government of India. S.N.B. and S.M. were supported by fellowships from the Council of Scientific and Industrial Research.