Isolation and characterization of the neutral leucine aminopeptidase (LapN) of tomato.
Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins. The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains. Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides. Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program. LapN was a single-copy gene encoding a rare-class transcript. A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A. Comparison of LAP-N with other plant LAPs identified 28 signature residues that classified LAP proteins as LAP-N or LAP-A like. Overexpression of a His(6)-LAP-N fusion protein in Escherichia coli demonstrated distinct differences in His(6)-LAP-N and His(6)-LAP-A activities. Similar to LapA, the LapN RNA encoded a precursor protein with a molecular mass of 60 kD. The 5-kD presequence had features similar to plastid transit peptides, and processing of the LAP-N presequence could generate the mature 55-kD LAP-N. Unlike LapA, the LapN transcript contained a second in-frame ATG, and utilization of this potential initiation codon would yield a 55-kD LAP-N protein. The localization of LAP-N could be controlled by the balance of translational initiation site utilization and LAP-N preprotein processing.
Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside 92521-0124, USA.
Chao-Jung
CJ
Tu
Sang-Youl
SY
Park
Linda L
LL
Walling
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D035264:Flowers / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D018547:Plant Stems / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D010940:Plant Proteins; D007931:Leucyl Aminopeptidase