Abstract
Mitochondrial tRNA import is widespread, but mechanistic insights of how tRNAs are translocated across mitochondrial membranes remain scarce. The parasitic protozoan T. brucei lacks mitochondrial tRNA genes. Consequently, it imports all organellar tRNAs from the cytosol. Here we investigated the connection between tRNA and protein translocation across the mitochondrial inner membrane. Trypanosomes have a single inner membrane protein translocase that consists of three heterooligomeric submodules, which all are required for import of matrix proteins. In vivo depletion of individual submodules shows that surprisingly only the integral membrane core module, including the protein import pore, but not the presequence-associated import motor are required for mitochondrial tRNA import. Thus we could uncouple import of matrix proteins from import of tRNAs even though both substrates are imported into the same mitochondrial subcompartment. This is reminiscent to the outer membrane where the main protein translocase but not on-going protein translocation is required for tRNA import. We also show that import of tRNAs across the outer and inner membranes are coupled to each other. Taken together, these data support the 'alternate import model', which states that tRNA and protein import while mechanistically independent use the same translocation pores but not at the same time.