| Protein turnover by the ubiquitin (Ub)/26S proteasome system (UPS) is an essential proteolytic pathway that selectively removes misfolded/abnormal and short-lived regulatory proteins in all eukaryotes. Attachment of a poly-Ub chain marks proteins for degradation by the 26S proteasome, a multi-subunit, self-compartmentalized, ATP-dependent protease. 26S proteasome activity is regulated by subunit composition, post-translational modifications, interacting proteins, and activating complexes. Products of the proteasome are short-peptides, which are further broken down into amino acids by a number of proteases, including tripeptidyl peptidase II (TPPII). My thesis is focused on understanding the UPS in Arabidopsis thaliana.;Previous studies of the plant proteasome identified the core constituents of the complex, but failed to identify many of the regulatory mechanisms. I have developed an affinity purification method for rapidly isolating the 26S proteasome from whole Arabidopsis seedlings. Biochemical analyses have proven this method to be highly specific and successful for purifying the intact complex. Mass spectrometric analyses of my preparations demonstrated the incorporation of most proteasome subunit paralogs, detected numerous post-translational modifications, and identified proteasome-interacting proteins.;Second, I characterized the proteasome regulatory particle non-ATPase subunit (RPN)-5 isoforms RPN5a and RPN5b by a variety of genetic and biochemical techniques. Whereas single rpn5b mutants appear wild-type, single rpn5a mutants display a host of defects. Both RPN5a and RPN5b could rescue the rpn5a-1 phenotype. However, abnormal phenotypes were generated by overexpression of each isoform. Collectively, the data suggest that its two paralogous genes in Arabidopsis have both redundant and unique roles in development.;Finally, I purified and characterized Arabidopsis TPPII, a protease downstream of the proteasome that helps degrade short-peptide products derived from the 26S proteasome into free amino acids. Two related species of 153- and 142-kDa are present in the purified preparations that are expressed from a single TPP2 gene. Protein sequencing demonstrated that the size difference of these species was caused by a C-terminal truncation. Mutants defective in TPP2 expression displayed no phenotypic abnormalities, suggesting that other intermediate exopeptidases assist in amino acid recycling. Taken together, the 26S proteasome and TPPII represent important routes for removing unwanted protein and recycling amino acids in plants. |
