Formyltetrahydrofolic acid :methionyl-tRNA transformylase was isolated from Saccharomyces cerevisiae mitochondria and used to prepare yeast mitochondrial [1Hlformylmethionyl-tRNA. This fMet-tRNA hybridizes with mitochondrial DNA but not with yeast nuclear or E. coli DNA. Unlabeled mitochondrial, but not extramitochondrial, tRNA competes in this reaction. tRNA was eluted from the hybrid and found to contain the label almost exclusively in fMet-tRNA. Yeast cytoplasmic fMettRNA formylated with Escherichia coli enzyme, and E. coli fMet-tRNA, do not hybridize with mitochondrial DNA. It is concluded that yeast mitochondrial tRNAfMet is a gene product of the mitochondrial genome.N-formylmethionyl-tRNA has been detected in yeast and rat liver mitochondria (1) but not in the soluble cell fractions. Takeishi et al. could demonstrate no formyl (H)4folate: methionyl-tRNA transformylase activity in yeast extracts, but were able to separate a methionyl-tRNA species that could be formylated by an Escherichia coli enzyme preparation (2, 3). This tRNA species accounted for a large portion of the methionine-acceptor activity of the whole cell tRNA and therefore was unlikely to be of mitochondrial origin. It has recently been identified, in the nonformylated form, as the initiator of extramitochondrial protein synthesis (4).Recently our laboratory has identified several yeast mitochondrial RNA species as gene products of mitochondrial DNA (5-7) as part of a study of the roles of the mitochondrial and nuclear genomes in mitochondrial biogenesis (8, 9). The known participation of N-formylmethionyl-tRNA in initiating protein synthesis in bacteria, combined with the similarity in the characteristics of the mitochondrial and the bacterial protein synthetic systems, prompted our examination of mitochondrial DNA as the source of the genetic information specifying the synthesis of this tRNA species. We describe in these experiments the isolation of a fraction that contains transformylase activity from yeast mitochondria and the evidence that the mitochondrial tRNAfMet is a gene product of the mitochondrial DNA.
METHODSA respiratory-sufficient haploid yeast strain, D243-2B-R1 (a, Iys-, ad1-p+), obtained from Prof. P. P. Slonimski, was used in this study. Yeast was grown in a medium containing 1% yeast extract, 2% bactopeptone, and 2% galactose, and collected in the late exponential phase of growth. Mitochondria were isolated by digestion of the cell wall with glusulase (Endo Laboratories), homogenization, and centrifugal separation (8). Mitochondrial DNA was isolated from DNasetreated mitochondria by lysis in 0.01 M Tris HCl, pH 7.5, containing 0.01 M EDTA and 1% sodium dodecylsulfate, deproteinization by chloroform-n-octanol 9:1, and passage through a hydroxylapatite column (7). The DNA was tested for purity by isopycnic CsCl ultracentrifugation in the analytical ultracentrifuge (10).Yeast nuclear and E. coli K 12 DNA were isolated by a similar method, except that the yeast nuclear DNA was further purified by hydroxylapatite chro...
