Show simple item record

dc.contributor.authorUhrig, Richard Glen
dc.contributor.otherQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))en
dc.date2007-09-26 15:57:52.216en
dc.date.accessioned2008-01-09T16:52:01Z
dc.date.available2008-01-09T16:52:01Z
dc.date.issued2008-01-09T16:52:01Z
dc.identifier.urihttp://hdl.handle.net/1974/969
dc.descriptionThesis (Master, Biology) -- Queen's University, 2007-09-26 15:57:52.216en
dc.description.abstractCo-immunoprecipitation (co-IP) followed by proteomic analysis was employed to examine the phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS) endosperm. Earlier studies suggested that immunologically unrelated 107-kDa plant-type and 118-kDa bacterial-type PEPCs (p107/PTPC and p118/BTPC, respectively) are subunits of an unusual ~910-kDa hetero-octameric Class-2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 since p118 quantitatively co-IP’d with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PTPC or BTPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation site characteristic of PTPCs, Pro-Q Diamond Phosphoprotein staining, immunoblotting with phospho-(Ser/Thr) Akt substrate IgG, and phosphate-affinity PAGE demonstrated that the co-IP’d p118 was significantly phosphorylated at unique Ser and/or Thr residue(s). The co-IP of p118 and p107 was not influenced by their phosphorylation status. As p118 phosphorylation appeared unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118’s in vivo phosphorylation. A third PEPC polypeptide of ~110-kDa (p110; RcPPC1) co-IP’d with p118 and p107 when depodded COS was used. Analysis of RcPpc1’s full-length cDNA sequence revealed p110’s identity with PTPCs, but that a pair of unique amino-acid substitutions occurs in its N-terminal sequence that may render p110 non-phosphorylatable in vivo. The plastidial pyruvate dehydrogenase complex (PDCpl) was identified as a novel PEPC interactor. Subcellular fractionation indicated that p118 and p107 are strictly cytosolic, but that PDCpl is targeted to both the cytosol and leucoplast of developing COS. Thus, a putative cytosolic metabolon involving PEPC and PDCpl could function to channel carbon from phosphoenolpyruvate to acetyl-CoA and/or to recycle CO2 from PDCpl to PEPC.en
dc.format.extent5773689 bytes
dc.format.mimetypeapplication/pdf
dc.languageenen
dc.language.isoenen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis 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.en
dc.subjectPEPCen
dc.subjectCo-IPen
dc.subjectFatty acid synthesisen
dc.subjectProteomicsen
dc.titleCo-immunoprecipitation analysis of the phosphoenolpyruvate carboxylase interactome of developing castor oil seedsen
dc.typeThesisen
dc.description.degreeMasteren
dc.contributor.supervisorPlaxton, William C.en
dc.contributor.departmentBiologyen


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record