Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves

Abstract

The constitutive and wound-inducible leucine aminopeptidases (LAP-N and LAP-A, respectively) of tomato encode 60-kDa proteins with 5-kDa presequences that resemble chloroplast-targeting peptides. Cell fractionation studies and immunoblot analyses of chloroplast and total proteins have suggested a dual location of the mature LAP-A proteins in the cytosol and the plastids. In this study, the subcellular localization of tomato LAPs was further investigated using in vitro chloroplast-targeting assays and immunocytochemical techniques at the light and TEM levels. In vitro-translated LAP-A1 and LAP-N preproteins were readily transported into pea chloroplasts and processed into mature proteins of 55 kDa indicating the presence of a functional chloroplast-targeting signal in the LAP-A1 and LAP-N protein precursors. In addition, a LAP polyclonal and a LAP-A-specific antisera were used to immunolocalize LAP proteins in leaves from healthy, wounded and methyl jasmonate (MeJA)-treated plants. Low levels of LAPs and/or LAP-like proteins were detected in leaves from unwounded plants. The LAP polyclonal antiserum, which detected LAP-A, LAP-N and LAP-like proteins, and the LAP-A specific antibodies, which detected only LAP-A, showed that LAP levels increased in leaf sections after wounding and MeJA treatments. LAP-A proteins were primarily detected within the chloroplasts of spongy and palisade mesophyll cells. The localization of LAP-A was distinct from the location of early wound-response proteins that are important in the biosynthesis of jasmonic acid or systemin and more similar to the late wound-response proteins with primary roles in defense. The importance of these findings relative to the potential roles of LAP-A in defense is discussed.

Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-induced Damage

“…However, due to the differences in their subcellular localizations, the peptidase/chaperone substrates of the Arabidopsis LAP1, which is located in the cytosol (33), are likely to be distinct from those of the plastid-localized LAP-A, LAP-N, and LAP2 (6,18). In addition, it is possible that the stress-induced LAP-A has a different set of peptidase/chaperone substrates from LAP-N and LAP2.…”

“…LAP-A resides within the plastid (18), which is a dynamic compartment subject to many cellular stresses during development and biotic/abiotic stresses that can result in protein damage and aggregation. In order to prevent the accumulation of misfolded proteins, cells express a wide range of molecular chaperones (19).…”

Leucine Aminopeptidase Regulates Defense and Wound Signaling in Tomato Downstream of Jasmonic Acid

“…Finally, exogenous application of JA to LapA-SI and wildtype plants showed that JA did not restore wound signaling in LapA-SI lines, indicating that LAP-A acted downstream of JA biosynthesis. Collectively, these data and the facts that LAP-A and Prosystemin are localized to distinct cellular compartments indicated that LAP-A is unlikely to influence the biogenesis of systemin (Narvá ez-Vá squez and Ryan, 2004;Narvá ez-Vá squez et al, 2007).…”

“…The LAP-A1 and LAP-A2 mature proteins are 99% identical and reside within the chloroplast (Matsui et al, 2006;Narvá ez-Vá squez et al, 2007). LAP-A1 and LAP-A2 are 77% identical with LAP-N, which is expressed at low levels constitutively (Tu et al, 2003).…”

A leucine aminopeptidase is involved in kinetoplast DNA segregation in Trypanosoma brucei

“…Members of the M1 family carry a canonical HEXXH motif in their active site, whereas the M17 family members lack this motif and require two metal ions per monomer for activity [31]. LAPs have diverse subcellular localizations; initially found in the cytosol, they have been subsequently encountered in chloroplasts [32], on bacterial surfaces [33], or teguments of parasitic helminths [34]. Moreover, in Escherichia coli LAPs bind DNA [35], while they are amongst secreted proteins in other bacteria such as Mycoplasma [33].…”