Localization and post-translational processing of the wound-induced leucine aminopeptidase proteins of tomato

Abstract

Leucine aminopeptidase (LAP) is induced wounding and bacterial pathogen infection in tomato. DNA blot analysis of XbaI-digested λlap genomic clones demonstrated that LapA1 and LapA2 cDNAs were encoded by two different LapA genes in the tomato genome. The coding and untranslated regions of LapA1 and LapA2 mRNAs shared more than 93% identity. The deduced amino acid sequences of LapA cDNA clones and in vitro translation of LapA1 mRNA indicated that LAP-A was synthesized as a 60-kDa precursor protein. The processing of a 60- kDa preLAP-A into the mature 55-kDa LAP-A was demonstrated in vivo by expression of the full-length LapA1 eDNA in insect cells. Sequencing of a single LAP-A form isolated from a two-dimensional polyacrylamide gel indicated that LAP-A proteins had two different N termini that were separated by two residues. The LAP-A presequence had features similar to chloroplast transit peptides. Comparison of LAP-A levels in chloroplast and total protein extracts from methyl jasmonate-treated leaves indicated that a small proportion of the LAP-A proteins was detected in the plastids. Inspection of the LAP-A presequence indicated the presence of a dibasic protease (Kex2/furin) processing site motif 6-8 residues upstream from the LAP-A N termini. Its potential role in LAP-A precursor biogenesis is discussed.