Formate transformylase

Our own involvement in this area of research came about in an indirect way. A major interest in the laboratory had been the role of vitamin Bja in methyl group transfer from N -methyl-H4-folate to homocysteine to yield methionine, a problem that was an extension of studies on the isolation of the vitamin coenzyme while in Horace A. Barker s laboratory in 1958. When the exciting report by Clark and Marcker appeared showing that W-formylmethionine-tRNA (fMet-rRNA) was the initiator or protein synthesis, it was a natural extension of our work to examine the formation of fMet-tRNA, since this reaction also involved both a one-carbon transfer from a reduced folate derivative and the amino acid methionine. Herbert Dickerman, a postdoctoral fellow in the laboratory at that time, was able to purify the transformylase enzyme from E. coU extracts that catalyzed eqn. (1) ... 

This claim is based on the following observations (1) Like that of their prokaryotic ancestors, mitochondrial translation uses fMet-tRNA (rather than the Met-tRNA used by cytoribosomes) as the initiator. (2) It can do so successfully because the transformylase which converts the Met-tRNA into its fMet derivative is mitochondrial in its localization. (3) The formation of the initiation complex can be monitored by the transfer of labeled formate (f ) to f Met to puromycin, resulting in its quantitative conversion to f Met-puro —a reaction that is restricted both in vitro and in vivo to the mitochondrial fraction and can be shown to go on with linear kinetics for extended periods. (4) Retention of f Met on nascent polypeptide chains is restricted to mitochondrial polyribosomes and can be used as a specific means for the identification and characterization of the latter. (5) Mitochondria, at least of the yeast species examined by us, appear to be deficient in both a deformylase capable of removing formate from fMet, whether free or on polypeptides, as well as in peptidases capable of removing either this component itself or small peptides from the N-terminal end. (6) Initiation by fMet is absent in p" mutants. In principle then, presence of formate as N-terminal fMet in a polypeptide provides an unambiguous means for its identification as having been synthesized on mitoribosomes. In practice, although feasible, as will be shown in the next section, this is difficult because it requires the prior... 

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