AN INTACT PROTEIN TRANSLOCATING MACHINERY IS REQUIRED FOR MITOCHONDRIAL IMPORT OF A YEAST CYTOPLASMIC TRANSFER-RNA

In Saccharomyces cerevisiae, one of the two cytoplasmic lysine-tRNA is oacceptors, tRNA(CUU)(Lys), is the only nuclear-coded tRNA species to be mitochondrially-imported. Using an in vitro assay of tRNA import, w e have studied if the mitochondrial protein translocation machinery is also involved in the mitochondrial targeting of this tRNA. As for pro tein import, tRNA import also requires the integrity of proteins expos ed onto the mitochondrial outer membrane and is dependent upon energy and membrane eletrochemical potential. Furthermore, partial ATP-deplet ion of the import system leads to stabilization of a translocation int ermediate containing the tRNA. In this case, the import of the tRNA ca n be completed after addition of a new source of energy, even without restoration of the membrane electrochemical potential and without furt her addition of cytoplasmic proteins. Outer and inner mitochondrial me mbrane receptors shown to participate in translocation of mitochondria l pre-proteins (MOM19 and MPI1/MIM44, respectively) are also important for import of the tRNA, in vitro as well as in vivo. In contrast, the outer membrane receptor MOM72 is dispensable for the import. Taken to gether, these results demonstrate that a functional mitochondrial prot ein translocation apparatus is required for the mitochondrial import o f the yeast tRNA(CUU)(Lys).