Proton-guided movements of tRNA within the Leishmania mitochondrial RNA import complex

Abstract

The RNA import complex (RIC) from the mitochondrion of the kinetoplastid protozoan Leishmania tropica contains two subunits that directly bind to import signals on two distinct subsets of tRNA and interact with each other allosterically. What happens to the tRNA subsequent to its loading on the complex is unknown. A third subunit-RIC9-has intrinsic affinity for both types of tRNA and is essential for import in vivo. Here we show that antibody against RIC9 inhibited the import of both types of tRNA into mitoplasts in vitro, but failed to inhibit the binding of these tRNAs to their respective receptors, indicating that RIC9 acts in a subsequent step. Using photoaffinity crosslinking-immunoprecipitation to detect translocation intermediates, it was observed that tRNA was transferred from its cognate receptor to RIC9, followed by translocation across the membrane and release as free tRNA in the inner compartment. Transfer required elevated temperatures and ATP, but ATP was substituted by acid pH. These tRNA movements were sensitive to uncouplers and inhibitors, suggesting distinct roles of the electrical and chemical components of the proton motive force generated by vectorial proton translocation accompanying ATP hydrolysis. By analysis of partially assembled complexes in L. tropica depleted of various subunits, and in vitro assembly assays, RIC9 was shown to make stable contacts with RIC8A, a tRNA receptor and RIC6, a membrane-embedded component. The results have implications for the mechanism of tRNA import.