In a significant fraction of the Escherichia coli cytosolic proteins, the N-terminal methionine residue incorporated during the translation initiation step is excised. The N-terminal methionine excision is catalyzed by methionylaminopeptidase (MAP). Previous studies have suggested that the action of this enzyme could depend mainly on the nature of the second amino acid residue in the polypeptide chain. In this study, to achieve a systematic analysis of the specificity of MAP action, each of the 20 amino acids was introduced at the penultimate position of methionyl-tRNA synthetase of E. coli and the extent of in vivo methionine excision was measured. To facilitate variant protein purification and N-terminal sequence determination, an expression shuttle vector based on protein fusion with (3-galactosidase was used. From our results, methionine excision catalyzed by MAP is shown to obey the following rule: the catalytic efficiency of MAP, and therefore the extent of cleavage, decreases in parallel with the increasing of the maximal side-chain length of the amino acid in the penultimate position. This molecular model accounts for the rate of N-terminal methionine excision in E. coli, as deduced from the analysis of 100 protein N-terminal sequences.Incorporation of a methionine residue at the N terminus of each nascent polypeptide makes part of the universal translation initiation signal, used by prokaryotes as well as eukaryotes. In prokaryotes, the methionyl moiety carried by the initiator tRNA is N-formylated prior to its incorporation. However, soluble proteins retaining a formylated N terminus do not represent a measurable fraction of total proteins in Escherichia coli (1, 2). Moreover, in a cytosolic extract of E. coli, only 40% of the polypeptidic chains retain an N-terminal methionine. Instead, about 50% display alanine, serine, or threonine at their N termini (3).These observations are accounted for by early posttranslational modifications of the polypeptides. The formyl group and methionine residue are removed sequentially, the deformylation step being more tightly coupled to the translation process than the methionine excision (4). The occurrence of two separate activities was established by the purification of the E. coli deformylase enzyme (2, 5-7) and, very recently, by the cloning of the methionyl-aminopeptidase (MAP) gene from both E. coli and Salmonella typhimurium (8, 9).Previous studies (2, 10) and, more recently, a survey (11) of published protein sequences have attempted to find out a rule for the conditional in vivo excision of the initiator methionine residue. However, the specificity of the removal of this residue by the E. coli MAP could never be systematically described, neither in vitro nor in vivo, nor had its biological relevance ever been substantiated.Experiments conducted in vitro on oligopeptides with purified MAP (8), in a eukaryotic cell-free expression system (12) or in vivo in yeast (11,13), suggest that the MAP specificity mainly depends on the nature of the second amino ...