The Proteomics of N-terminal Methionine Cleavage

Thierry Meinnel,Richard C. Holz,Frédéric Frottin,Philippe Peynot,Carmela Giglione,Aude Martinez,Sanghamitra Mitra

doi:10.1074/mcp.m600225-mcp200

Abstract

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1'), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1' were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2', P3', and P4' strongly slowed the reaction, and some proteins with Val and Thr at P1' could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought.

Highlights

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision
We found that proteins with Thr and Val at P1Ј were poor substrates of MAP1 and MAP2, frequently resisting N-terminal methionine excision (NME) cleavage, and that the P2Ј and positions 3 (P2Ј) and 4 (P3Ј) positions had a strong effect on specificity for both methionine aminopeptidase (MAP) types
In a previous analysis of the E. coli NME machinery, we showed that MAP was the primary determinant of the nature of the N terminus of a protein and that the most sensitive position for EcMAP1 action was position P1Ј (Fig. 3)

Summary

Introduction

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. Certain residues at P2؅, P3؅, and P4؅ strongly slowed the reaction, and some proteins with Val and Thr at P1؅ could not undergo Met cleavage These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. Edman degradation sequencing of two reporter proteins in E. coli was used to further the analysis [17, 18] The authors of these studies suggested that the process was statistical rather than stochastic and that cleavage efficiency was correlated with side-chain length or gyration radius as defined by Levitt [19] at P1Ј [20]. These findings form the basis of our current understanding of the process (for a review, see Ref. 1; see Fig. 1 in Ref. 21), which is used for bioinformatics

Objectives

Results

Conclusion