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Molecular and Cellular Biology, August 2002, p. 5434-5442, Vol. 22, No. 15
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.15.5434-5442.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Vaidyanathan Ramesh, Caroline Köhrer, and Uttam L. RajBhandary*

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 31 January 2002/ Returned for modification 8 March 2002/ Accepted 30 April 2002

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.

* Corresponding author. Mailing address: Department of Biology, 68-671 MIT, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-4702. Fax: (617) 252-1556. E-mail: bhandary{at}mit.edu.

Molecular and Cellular Biology, August 2002, p. 5434-5442, Vol. 22, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.15.5434-5442.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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