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Title: A NOVEL C-TERMINAL PROTEOLYTIC PROCESSING OF CYTOSOLIC PYRUVATE KINASE, ITS PHOSPHORYLATION AND DEGRADATION BY THE PROTEASOME IN DEVELOPING SOYBEAN SEEDS

Author
item TANG, GUO-QING - NC STATE UNIV
item Hardin, Shane
item DEWEY, RALPH - NC STATE UNIV
item Huber, Steven

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/9/2003
Publication Date: 4/1/2003
Citation: Tang, G., Hardin, S.C., Dewey, R., Huber, S.C. 2003. A novel c-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds. Plant Journal. 34:77-95.

Interpretive Summary: Developing soybean seeds use sucrose and amino acids, obtained from the maternal plant, to produce the storage protein and oil that determine the value of the crop. Hence it is important to understand the biological mechanisms that control the metabolic pathways leading to biosynthesis of storage products during seed development. Sucrose is metabolized by a pathway known as glycolysis to produce small carbon skeletons that can be used either for protein or oil synthesis. We speculated that the enzyme "cytosolic pyruvate kinase" may act at the branch-point controlling the flow of carbon into protein versus oil. However, little is known about the biological mechanisms controlling pyruvate kinase. Results obtained suggest that this enzyme can be modified in two different ways after it is synthesized (referred to as post-translational modification). First, a slightly smaller version of the protein is produced, apparently by removal of a portion of the end of the protein, known as the carboxy-terminus. This form of the protein accumulates during seed development, and may have different regulatory properties. Second, the pyruvate kinase protein can be modified by covalent attachment of a phosphate molecule in a process known as protein phosphorylation. Phosphate was shown to be attached to the protein at two specific serine residues. Surprisingly, the effect of phosphorylation seems to be to target the pyruvate kinase protein for proteolytic degradation. These results add to our knowledge of the biological mechanisms that may control seed metabolism and composition.

Technical Abstract: Cytosolic pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase; EC 2.7.1.40) is a key enzyme regulating glycolysis, but the post-translational regulation of this enzyme is poorly understood in higher plants. Sequence analysis of the soybean seed enzyme suggested potential for two phosphorylation sites: site-1 (FVRKGS220DLVN) and site-2 (VLTRGG S407TAKL). Sequence - and phosphorylation - state specific antipeptide antibodies established that cytosolic pyruvate kinase (PyrKinc) is phosphorylated at both sites in vivo. However, by SDS-PAGE, the phosphorylated polypeptides were smaller (20 to 51-kDa) than the full-length (55-kDa). Biochemical separations of seed proteins by size exclusion chromatography and sucrose-density gradient centrifugation revealed that the phosphorylated polypeptides were associated with 26S proteasomes. The 26S proteasome particle in the developing soybean seed was determined to be ~ 1900-kDa. In vitro, the 26S proteasome degraded the associated pyruvate kinase polypeptides and this was blocked by proteasome-specific inhibitors such as MG132 and NLVS. By immunoprecipitation, we found that some of the phosphorylated pyruvate kinase was conjugated to ubiquitin and shifted to high molecular mass forms in vivo. Moreover, recombinant wild type PyrKinc was ubiquitinated in vitro to a much greater extent than the S220A and S407A mutant proteins, suggesting a link between phosphorylation and ubiquitination. In addition, during seed development, a progressive accumulation of a C-terminally truncated ~ 51-kDa polypeptide was observed that was paralleled by a loss of the full length 55-kDa polypeptide. Interestingly, the C-terminal 51-kDa truncation showed not only pyruvate kinase activity but also apparently activation by aspartate. Collectively, the results suggest that there are two pathways for pyruvate kinase modification at the post-translational level. One involves the partial C-terminal truncation to generate a 51-kDa pyruvate kinase subunit which might have altered regulatory properties. The other involves phosphorylation and ubiquitin-conjugation that targets the protein to the 26S proteasome for complete degradation. This is one of the first examples of degradation of a plant metabolic enzyme via the ubiquitin / 26S proteasome pathway.