Dual specificities of the glyoxysomal/peroxisomal processing protease Deg15 in higher plants

Abstract

Glyoxysomes are a subclass of peroxisomes involved in lipid mobilization. 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. The cleavage site typically contains a Cys at P1 or P2. We purified the glyoxysomal processing protease (GPP) from the fat-storing cotyledons of watermelon (Citrullus vulgaris) by column chromatography, preparative native isoelectric focusing, and 2D PAGE. The GPP appears in two forms, a 72-kDa monomer and a 144-kDa dimer, which are in equilibrium with one another. The equilibrium is shifted on Ca 2؉ removal toward the monomer and on Ca 2؉ addition toward the dimer. The monomer is a general degrading protease and is activated by denatured proteins. The dimer constitutes the processing protease because the substrate specificity proven for the monomer (⌽-Arg/Lys2) is different from the processing substrate specificity (Cys-Xxx2/Xxx-Cys2) found with the mixture of monomer and dimer. The Arabidopsis genome analysis disclosed three proteases predicted to be in peroxisomes, a Deg-protease, a pitrilysin-like metallopeptidase, and a Lonprotease. Specific antibodies against the peroxisomal Degprotease from Arabidopsis (Deg15) identify the watermelon GPP as a Deg15. A knockout mutation in the DEG15 gene of Arabidopsis (At1g28320) prevents processing of the glyoxysomal malate dehydrogenase precursor to the mature form. Thus, the GPP/Deg15 belongs to a group of trypsin-like serine proteases with Escherichia coli DegP as a prototype. Nevertheless, the GPP/Deg15 possesses specific characteristics and is therefore a new subgroup within the Deg proteases.Arabidopsis thaliana ͉ Ca 2ϩ signal ͉ Citrullus vulgaris ͉ monomer/dimer equilibrium N umerous matrix enzymes have to be imported from the cytosol into peroxisomes, in plants especially into glyoxysomes for seed storage oil mobilization or into leaf peroxisomes for photorespiration. The majority of these enzymes are imported in their mature form and targeted by a C-terminal SKL designated peroxisomal targeting signal 1 (PTS1) (1). In a few matrix enzymes, the peroxisomal targeting signal 2 (PTS2) with the consensus RL-X5-HL is located in the N-terminal 30 to 50 amino acids of the protein (2). In plants, these are four enzymes of the glyoxylate cycle and ␤-oxidation of fatty acids: glyoxysomal malate dehydrogenase (gMDH), glyoxysomal citrate synthase (gCS), acyl-CoA oxidase, and thiolase. In mammals, three enzymes with a PTS2 have been identified: thiolase, alkyl-DHAP synthase, and phytanoyl-CoA hydroxylase. In higher eukaryotes, such as plants and mammals, the PTS2 is removed on import; a Cys is consistently found near the cleavage site [supporting information (SI) Table 2]. In lower eukaryotes, such as yeasts, a PTS2 is present in the N terminus of the mature subunit of thiolase and amine oxidase (2) (SI Table 2). The Cys in position P2 is required for processing the presequences of gCS and gMDH in pumpkin; deletion of the...

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…The PTS2 sequence is usually cleaved from the cargo protein after import into the peroxisome, a process that is not observed for PTS1-containing proteins (Tanaka et al, 2008). The protease that performs this cleavage has been identified as DEG15 in plants (Helm et al, 2007) and tysnd1 in mammals (Kurochkin et al, 2007). deg15 mutants have very mild peroxisome deficiency defects, suggesting that processing is not required for import (Lingard and Bartel, 2009).…”

Peroxisome-associated matrix protein degradation in Arabidopsis

“…3B), indicating that MLS turnover was also PEX4 dependent. We also monitored thiolase, a fatty acid ␤-oxidation protein with a type 2 peroxisome targeting signal (PTS2) that is cleaved after import into the peroxisome matrix (29), which was most abundant at 3 days and gradually declined to a lower basal level as wild-type seedling matured (21) (Fig. 3B).…”

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

“…PEX7 recognizes proteins bearing a PTS2 (Marzioch et al, 1994), a nine-amino acid sequence embedded in an ;30-amino acid N-terminal presequence. This presequence is removed upon peroxisome import in mammals and plants (Swinkels et al, 1991;Helm et al, 2007). After cargo translocation into the organelle, PEX5 and PEX7 are returned to the cytosol for reuse (Collins et al, 2000;Dammai and Subramani, 2001;Nair et al, 2004).…”

“…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 .…”