Import of the Peroxisomal Targeting Signal Type 2 Protein 3-Ketoacyl-Coenzyme A Thiolase into Glyoxysomes

Abstract

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.The enzyme 3-ketoacyl-CoA-thiolase (EC 2.3.1.16; thiolase) is a homodimer that catalyzes the terminal reaction of fatty acid ␤-oxidation in eukaryotes. In many organisms, thiolase is located in both the mitochondrial matrix and the peroxisome, but in plants, it appears to be localized exclusively to peroxisomes, including the specialized type of plant peroxisome, the glyoxysome (Kindl, 1992). No thiolase activity has been localized to plant mitochondria. Like all peroxisomal matrix proteins, thiolase is synthesized on free polyribosomes in the cytosol and posttranslationally imported into the organelle (Johnson and Olsen, 2001).Most peroxisomal matrix proteins use a carboxyterminal tripeptide targeting signal, termed a peroxisomal targeting signal type 1 (PTS1; Gould et al., 1989). In contrast, thiolase is directed to the organelle by a distinct signal, the peroxisomal targeting signal type 2 (PTS2; Gietl, 1990;Osumi et al., 1991;Swinkels et al., 1991; Kato et al., 1996). The PTS2 is nonapeptide signal with the consensus sequence (R) X 6 (H/ Q)(L/A/F); it is located near the amino terminus of the peroxisomal protein (Flynn et al., 1998;Olsen, 1998). In addition to thiolase, other plant proteins that contain a PTS2 include citrate synthase (Kato et al., 1996), malate dehydrogenase (Gietl et al., 1994), amine oxidase (Faber et al., 1995), and at least one isozyme of Asp aminotransferase (Gebhardt et al., 1998). Plants appear to have more PTS2 proteins than other organisms.Relatively few proteins follow the PTS2 pathway, but their import is necessary for the survival of the organ...

A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production

“…PhKAT1 is predicted to encode a protein of 462 amino acids containing three conserved thiolase signatures (Figure S2). Typically for thiolases, PhKAT1 contains a peroxisomal targeting signal 2 (Gietl, 1990; Johnson and Olsen, 2003; Reumann, 2004; Carrie et al. , 2007) at the N‐terminus (Figure S2).…”

“…The petunia 3-ketoacyl-CoA thiolase protein is localised to peroxisomes in planta PhKAT1 is predicted to encode a protein of 462 amino acids containing three conserved thiolase signatures ( Figure S2). Typically for thiolases, PhKAT1 contains a peroxisomal targeting signal 2 (Gietl, 1990;Johnson and Olsen, 2003;Reumann, 2004;Carrie et al, 2007) at the N-terminus ( Figure S2). To verify whether PhKAT1 is localised to the peroxisomes in planta, we constructed a PhKAT1-GFP fusion protein and expressed it in petunia petals by means of Agrobacterium-mediated transient transformation (Shang et al, 2007).…”

Diversity in subcellular targeting of the PP2A B′η subfamily members

“…This also implies that proteins can be correctly folded and have their cofactors or interacting proteins bound, before the whole complex is transferred into the peroxisomes as a cargo. Examples of proteins lacking a peroxisomal targeting signal, but transported into these organelles by binding to other proteins possessing such signals ("piggy-backing") have been described (Johnson and Olsen 2003;Brocard and Hartig 2006). Possibly, the BЈ may bind a catalytic subunit C and scaVolding subunit A, none of which have a peroxisomal targeting signal (SUBAII, Heazlewood et al 2005), before being transported into the peroxisome as a holoenzyme.…”

Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria

“…However, the thiolase is question is a type I enzyme, while in mammals it is the mitochondrial type II ACATs that have been implicated in isoleucine catabolism (Pereto et al 2005). The KAT2 thiolase encoded by At2g33150 has a predicted type 2 peroxisomal targeting sequence (PTS2) conforming to the consensus R-(X)6-H/Q-A/L/F with a downstream cysteine residue required for proteolytic cleavage (Baker and Sparkes 2005), and is imported into peroxisomes in vitro (Johnson and Olsen 2003). At2g33150 is well known to be essential for peroxisomal b-oxidation (Germain et al 2001).…”