Characterization and Cloning of Tripeptidyl Peptidase II from the Fruit Fly, Drosophila melanogaster

Renn, Susan C. P.; Tomkinson, Birgitta; Taghert, Paul H.

doi:10.1074/jbc.273.30.19173

Cited by 34 publications

(29 citation statements)

References 42 publications

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“…Following SDS-PAGE, these two species were analyzed by matrix-assisted laserdesorption ionization time of flight (MALDI-TOF)-mass spectrometry (MS) fingerprinting following digestion of each with trypsin. The molecular masses of at least five peptides from each species matched the theoretical tryptic map of a single Arabidopsis open reading frame At4g20850 (now designated TPPII for the protein and TPP2 for the genomic locus), which has strong sequence similarity to animal and Drosophila TPPII (Renn et al, 1998). Even though two TPPII species of 153 and 142 kD were evident by SDS-PAGE, only one TPP2 coding sequence could be found in the near-complete Arabidopsis genomic sequence.…”

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 97%

“…Similar to other TPPII proteins, the Arabidopsis form also has an approximately 200-amino acid insertion (residues 152-369) between the catalytic Asp and His residues that appears to distinguish these peptidases from other members of the subtilisin superfamily (Renn et al, 1998;Tomkinson et al, 2002). Like its close relatives in other species, Arabidopsis TPPII also has an extended C-terminal domain (residues 1,195-1,380) that is highly variable with numerous alignment gaps as compared to its orthologs (Fig.…”

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 97%

“…The diamonds identify the conserved Asp, His, and Ser residues that form the catalytic triad. The circle indicates the Asn that stabilizes the tetrahedral intermediate (Renn et al, 1998). The site of the T-DNA insertion for the tpp2-1 mutant is indicated by the asterisk.…”

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 99%

“…Whereas the Ser protease inhibitor PMSF attenuated its activity, the Cys (E64c, leupeptin) and metalloprotease inhibitors (EDTA) were without an effect. Mammalian and Drosophila TPPIIs are sensitive to thiol-reactive compounds, mainly through their reaction with a positionally conserved Cys (residue 760 in Drosophila TPPII; Renn et al, 1998). This Cys has been substituted for an Ala in Arabidopsis TPPII, Identical and similar amino acids are shown in black and gray boxes, respectively.…”

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 99%

“…Like TPPII preparations from animals, the Arabidopsis peptidase was effectively quenched by the inhibitors AAF-chloromethylketone (CMK) and butabindide (Fig. 4, A and B), thus placing the enzyme in the TPPII clade of the subtilisin superfamily (Siezen and Leunissen, 1997;Renn et al, 1998). Butabindide was specifically designed as a competitive inhibitor of TPPII, using the rat peptidase for the assay (Rose et al, 1996).…”

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 99%

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Tripeptidyl Peptidase II. An Oligomeric Protease Complex from Arabidopsis

et al. 2005

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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...

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Section: Purification Of Tppii From Arabidopsismentioning

confidence: 97%

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 97%

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 99%

Section: Purification Of Tppii From Arabidopsismentioning

confidence: 99%