Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA

Protein synthesis in eukaryotic organelles such as mitochondria and chlorop lasts is widely believed to require a formylated initiator methionyl tRNA ( fmet-tRNA(fMet)) for initiation. Here we show that initiation of protein sy nthesis in yeast mitochondria can occur without formylation of the initiato r methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N- 10-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have stud ied yeast mutants carrying chromosomal disruptions of the genes encoding th e mitochondrial C-1-tetrahydrofolate (C-1-THP) synthase (MIS1), necessary f or synthesis of IO-formyl-THF, and the methionyl-tRNA formyltransferase (op en reading frame YBL013W; designated FMT1). A direct analysis of mitochondr ial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMe t), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vi vo of the mitochondrial initiator Met-tRNA in these strains. In contrast, t he initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exh ibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal fr equencies of generation of petite colonies. The only growth phenotype obser ved was a longer lag time during growth an nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion s train.