“Ping-Pong” Interactions between Mitochondrial tRNA Import Receptors within a Multiprotein Complex

Abstract

ABSTRACT The mitochondrial genomes of a wide variety of species contain an insufficient number of functional tRNA genes, and translation of mitochondrial mRNAs is sustained by import of nucleus-encoded tRNAs. In Leishmania , transfer of tRNAs across the inner membrane can be regulated by positive and negative interactions between them. To define the factors involved in such interactions, a large multisubunit complex (molecular mass, ∼640 kDa) from the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania , consisting of ∼130-Å particles, was isolated. The complex, when incorporated into phospholipid vesicles, induced specific, ATP- and proton motive force-dependent transfer of Leishmania tRNA Tyr as well as of oligoribonucleotides containing the import signal YGGYAGAGC. Moreover, allosteric interactions between tRNA Tyr and tRNA Ile were observed in the RNA import complex-reconstituted system, indicating the presence of primary and secondary tRNA binding sites within the complex. By a combination of antibody inhibition, photochemical cross-linking, and immunoprecipitation, it was shown that binding of tRNA Ile to a 21-kDa component of the complex is dependent upon tRNA Tyr , while binding of tRNA Tyr to a 45-kDa component is inhibited by tRNA Ile . This “ping-pong” mechanism may be an effective means to maintain a balanced tRNA pool for mitochondrial translation.