| The ubiquitin/26S proteasome pathway is one of the most important proteolytic pathways in eukaryotes, controlling the turnover of many short-lived regulators. A polyubiquitinated target protein is sent to the 26S proteasome for breakdown into peptide fragments, while the ubiquitins are released by deubiquitinating enzymes (DUBs) for reuse. Downstream proteases, such as tripeptidyl peptidase II (TPPII), further process the peptides to release amino acids.; Prior to this study, little was known about the biochemistry and regulation of the 26S proteasome in plants. First, I developed a protocol to purify the 26S proteasome from Arabidopsis thaliana. I showed that the plant complex has biochemical properties and inhibitor sensitivities similar to those previously reported for homologs from other eukaryotes. Characterization of this complex identified most core subunits and detected the products from many paralogous genes, indicating that multiple 26S proteasome isoforms exist in plants.; Second, I purified and characterized Arabidopsis TPPII, which digests the peptides into tripeptides. It exists as a soluble ~5 to 9-MDa complex assembled from two polypeptides that are derived from a single gene, with the smaller form resulting from a C-terminal truncation of larger form. Inhibitor studies confirmed TPPII as a serine protease of the subtilisin superfamily. No obvious phenotypes were observed in tpp2-1 mutant plants, suggesting that other peptidases besides TPPII are involved in amino acid recycling.; Last, I studied two Arabidopsis regulatory DUBs, ubiquitin carboxyl-terminal hydrolases, UCH1 and 2. Plants overexpressing UCH1 ( 35S-UCH1) displayed epinastic leaves, decreased intemode elongation and increased shoot apical dominance, while the uch1-1 uch2-1 mutants resulted in hyponastic leaves and increased shoot apical dominance. These defects in auxin signaling were further supported by the synergistic effects of 35S-UCH1 when combined with the auxin resistant mutants axr1-3 or axr2-1. The products of an AUX/IAA reporter transgene, HS AXR3NT-GUS, were stabilized in 35S-UCHI plants, and destabilized in uch1-1 uch2-1 plants. These results suggest that UCH1 and 2 function as regulators of auxin signaling by affecting the ubiquitination state of AUX/IAA proteins. Taken together, this work provides the first detailed characterization of important proteases associated with the Ub/26S proteasome system in Arabidopsis. |
