Tripeptidyl Peptidase II. An Oligomeric Protease Complex from Arabidopsis

Abstract

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo-and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5-to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling.Proteolysis serves a variety of essential functions, including the elimination of misfolded or damaged proteins, the precise removal of regulatory proteins, and the maintenance of free amino acid pools needed for continual protein synthesis (Vierstra, 1996;Tomkinson, 1999). To facilitate this breakdown, plants and animals have evolved several proteolytic mechanisms for each subcellular compartment. In the vacuole/lysosome, proteins are catabolized by a variety of proteases and peptidases following their delivery to this hydrolytic compartment via endocytic and autophagic mechanisms (Thompson and Vierstra, 2005). For nuclear and cytoplasmic proteins, as well as abnormal polypeptides transported in a retrograde fashion from the endoplasmic reticulum to the cytoplasm, a major route involves ubiquitin (Ub) and the 26S proteasome (Smalle and Vierstra, 2004). Here, proteins destined for degradation are selectively tagged by the covalent attachment of multiple Ubs.These ubiquitinated proteins are then recognized and cleaved into smaller fragments by the 26S proteasome, a self-compartmentalized ATP-dependent protease complex with broad substrate and cleavage specificity.Products of the 26S proteasome are predominantly peptides 6 to 12 amino acids in length (Wenzel et al., 1994;Kisselev et al., 1999;Voges et al., 1999). Complete recycling requires further cleavage of these peptides by intermediate...

Evidence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway

“…Aminopeptidase (bestatin) and Asp protease (pepstatin) inhibitors did not have any effect on this activity either (Table 3). These results are in accordance with previous descriptions of the Arabidopsis TPPII protease (30) and confirm the assumption that the AAF-AMC-like activity measured in these pools was due to TPPII. Fig.…”

“…1A). This is in agreement with a previous report showing that Arabidopsis TPPII is a large oligomeric 5-9-MDa complex (30). A TOP-like activity was measured using the specific fluorogenic substrate Mcc-PLGPK-Dnp.…”

“…Although not essential for the generation of antigenic peptides in antigen processing, it was supposed to be necessary for efficient protein turnover (49,50). This protease was recently purified from Arabidopsis seedlings and characterized (30). In this study, we confirmed the presence of TPPII in the leaves of Arabidopsis plants and demonstrated its role in the catabolism of oxidatively damaged proteins.…”

“…This characterization was done using classical enzymatic activity measurements with the help of specific inhibitors and effectors, by a proteomics approach, and by measuring gene expression at the transcript level. Besides the 20 S proteasome, which was previously identified in different plant species (19,20), this work provides new insights into the functioning of peptidases previously identified in Arabidopsis-like TPPII (30) and LAP (48). In addition, we report the occurrence of proteases that have not been described in the plant kingdom until now, like TOP.…”

Diversity of Serine Hydrolase Activities of Unchallenged and Botrytis-infected Arabidopsis thaliana

“…ARA12 (SBT1.7) is a secreted subtilase required for the release of mucilage to break the seed coat during germination but with unknown function in leaves (43,44). TPP2 (tripeptidyl peptidase-II, SBT6.2) is a large oligomeric cytosolic protease that degrades peptides produced by the proteasome (45). Acylamino acid-releasing enzyme acts as a tetrameric protein complex in the chloroplast stroma and regulates the turnover of N-acylated proteins through the hydrolysis of the N-terminal N ␣ -acylated amino acids (46).…”