Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branchpoint of plant C-metabolism. Plant genomes encode several closely related plant-type PEPC (PTPC) isozymes, and a distantly related bacterial-type PEPC (BTPC). Two physically and kinetically distinct oligomeric classes of PEPC occur in the endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of 107-kDa PTPC subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between Class-1 PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. BTPC functions as a catalytic and regulatory subunit of the allosterically-desensitized Class-2 PEPC, hypothesized to support PEP-flux to malate for leucoplast fatty acid synthesis. Previous studies established that BTPC: (i) subunits of COS Class-2 PEPC are phosphorylated at multiple sites in vivo and (ii) phosphorylation at Ser425 provides a new tier of enzyme control in developing COS. LC MS/MS and LTQ-FT MS identified Thr4 and Ser451 as additional in vivo phosphorylation sites of immunopurified COS BTPC (corresponding to acidophilic and basophilic protein kinase consensus sequences, respectively). Immunoblots probed with a phosphorylation-site specific antibody raised against a synthetic phosphopeptide indicated that Ser451 phosphorylation is promoted during seed development, becoming maximal in stage VII (full cotyledon) COS. Although several pThr4 containing BTPC peptides were non-immunogenic, the collective results indicate that Thr4 is also phosphorylated in vivo. Kinetic effects of each phosphorylation site were examined using phospho-mimetic mutants of heterologously expressed COS BTPC. BTPC’s phosphorylation at Ser451 appears to be inhibitory, as reflected by significantly increased
Km(PEP) values, and reduced I50(malate) and I50(Asp) values of a S451D mutant. By contrast, kinetic characterization of a T4D phosphomimetic mutant indicated that Thr4 phosphorylation is not regulatory in nature. However, Thr4 exists in a conserved forkhead-associated (FHA) binding domain (pTXXD) that has received considerable prominence as a phospho-Thr dependent protein interaction module. These results further our understanding of multisite phosphorylation of BTPC in developing COS and its possible contribution to the control of Class-2 PEPC activity. / Thesis (Master, Biology) -- Queen's University, 2011-08-22 19:05:03.077
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/6662 |
| Date | 23 August 2011 |
| Creators | DALZIEL, Katie |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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