The DEG15 Serine Protease Cleaves Peroxisomal Targeting Signal 2-Containing Proteins in Arabidopsis

Abstract

Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. Recently, candidates for the responsible processing protease were identified from plants (DEG15) and mammals (TYSND1). We demonstrate that plants lacking DEG15 show an expressed phenotype potentially linked to reduced b-oxidation, indicating the impact of protein processing on peroxisomal functions in higher eukaryotes. Mutational analysis of Arabidopsis (Arabidopsis thaliana) DEG15 revealed that conserved histidine, aspartic acid, and serine residues are essential for the proteolytic activity of this enzyme in vitro. This indicates that DEG15 and related enzymes are trypsin-like serine endopeptidases. Deletion of a plant-specific stretch present in the protease domain diminished, but did not abolish, the proteolytic activity of DEG15 against the PTS2-containing glyoxysomal malate dehydrogenase. Fluorescence microscopy showed that a DEG15-green fluorescent protein fusion construct is targeted to peroxisomes in planta. In vivo studies with isolated homozygous deg15 knockout mutants and complemented mutant lines suggest that this enzyme mediates general processing of PTS2-containing proteins.

E3 ubiquitin ligase SP1 regulates peroxisome biogenesis in Arabidopsis

“…To assess the impact of sp1 on peroxisome protein import in pex14-2 plants further, we also performed immunoblot analysis of a PTS2-containing protein, 3-ketoacyl-CoA thiolase (thiolase), whose precursors are retained for a longer time in mutants defective in PTS2 protein import than in wild-type plants (17). After PTS2-containing proteins are delivered into the matrix, the N-terminal PTS2 peptides are removed by proteases such as DEG15 (46,47). By evaluating PTS2 protein processing, we could indirectly assess the import of PTS2-containing proteins or the import of PTS1-containing proteases such as DEG15 that are required for PTS2 processing.…”

Disrupting Autophagy Restores Peroxisome Function to anArabidopsis lon2Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

“…All mutants were in the Col-0 accession. lon2-2/SALK_043857 ), deg15-1/SALK_007184 (Schuhmann et al, 2008;), atg2-1/SALK_076727 (Yoshimoto et al, 2009), atg7-2/GABI_655B06 (Chung et al, 2010), and atg7-3/ SAIL_11_H07 (Lai et al, 2011;Wang et al, 2011) were previously described and were from the ABRC. lon2-2 35S:PTS2-GFP ) and pex7-2 (Ramón and Bartel, 2010) were previously described.…”

“…PMDH is expressed as a precursor (p); the PTS2 region is cleaved to a mature (m) form in the peroxisome. pex7-2 is a control displaying reduced PMDH processing because of reduced matrix protein import (Ramón and Bartel, 2010); deg15-1 displays reduced processing because it is a null allele of the PTS2 processing protease (Schuhmann et al, 2008;. Membranes from duplicate gels were serially probed with the indicated antibodies to obtain the top two and bottom three panels.…”

“…DEG15 processes PTS2 proteins into their mature forms by removing the PTS2-containing N-terminal region (Helm et al, 2007;Schuhmann et al, 2008), but DEG15 is not required for ICL or MLS degradation . Similarly, the peroxisomal M16 metalloprotease PXM16 is not required for degradation of glyoxylate cycle enzymes; pxm16 and lon2 pxm16 mutants efficiently degrade ICL and MLS .…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…After delivery, the PTS1 region is retained whereas the approximately 30-amino acid N-terminal region of plant PTS2 proteins is cleaved by the protease DEG15 ( Fig. 3; Helm et al, 2007;Schuhmann et al, 2008). Although plants, yeast, and mammals use both PTS1 and PTS2 systems, nematodes and fruit flies lack PTS2 proteins (Gurvitz et al, 2000;Motley et al, 2000;Faust et al, 2012).…”