Expression of Escherichia coli methionyl-tRNA formyltransferase in Saccharomyces cerevisiae leads to formylation of the cytoplasmic initiator tRNA and possibly to initiation of protein synthesis with formylmethionine.
Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.
Ramesh
Vaidyanathan
V
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Köhrer
Caroline
C
RajBhandary
Uttam L
UL
D000581:Amidohydrolases; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005561:Formates / Q000378:metabolism* / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D019877:Hydroxymethyl and Formyl Transferases / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D009239:N-Formylmethionine / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational / Q000502:physiology; D010641:Phenotype; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*
D001426:Bacterial Proteins; D005561:Formates; D012358:RNA, Transfer, Met; D012269:Ribosomal Proteins; C429752:ribosomal protein 3; C030544:formic acid; D009239:N-Formylmethionine; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase