Role of an RNA-binding protein in import of tRNA into Leishmania mitochondria

Abstract

Nuclear-encoded cytoplasmic tRNAs are imported into the mitochondria of kinetoplastid protozoa by an unknown mechanism. In a Leishmania in organello system, ATP-dependent import of a cloned, unspliced tRNATyr(GUA) transcript was demonstrated by protection from ribonuclease, whereas import of a tRNA^Gln(CUG) transcript was much less efficient. Specific binding of tRNA^Tyr to two mitochondrial surface proteins of 15 and 22 kilodaltons was observed. Tubulin antisense-binding protein (TAB), the 15-kilodaton species, was purified to apparent homogeneity by RNA affinity chromatography. TAB forms stable complexes with the D stem-loop region of tRNA^Tyr. Immunocytochemical and cell fractionation experiments, combined with limited proteolysis, suggested the association of TAB with the outer mitochondrial membrane. Importantly, anti-TAB antibody specifically inhibited binding as well as import of tRNA^Tyr and of a synthetic structural homolog. These results support the role of TAB as a membrane-bound receptor or carrier for RNA import into Leishmania mitochondria.