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Post-translational regulation of Arabidopsis thaliana starch synthase 2

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dc.contributor.advisor Emes, Michael
dc.contributor.advisor Tetlow, Ian
dc.contributor.author Patterson, Jenelle
dc.date.accessioned 2018-04-10T15:12:31Z
dc.date.available 2018-04-10T15:12:31Z
dc.date.copyright 2018-02
dc.date.created 2018-02-26
dc.date.issued 2018-04-10
dc.identifier.uri http://hdl.handle.net/10214/12598
dc.description.abstract Starch is the main carbohydrate storage molecule in plants. In photosynthetic tissues, it is produced during the light-period and degraded at night to support cellular respiration. Starch is synthesized by the coordinated actions of multiple enzyme classes: starch synthases (SS), starch branching enzymes (SBE) and debranching enzymes (DBE), each consisting of several isoforms. Starch synthase 2 (SS2) is important in starch synthesis, elongating intermediate-length glucan chains, and its loss results in distorted starch granules with altered physiochemical properties. Despite its importance in starch biosynthesis, the regulation of SS2 is poorly understood. In this study, post-translational mechanisms that potentially regulate SS2 were identified using a combination of in silico, in vitro, and in vivo analyses. The SS2 N-terminal region, comprised of the first 185 amino acids of the mature protein sequence, is highly variable between species and appeared to be intrinsically disordered. Intrinsic disorder in proteins is often correlated with protein phosphorylation and protein-interactions. Recombinant SS2 formed homodimers that required the N-terminal region, but N-terminal peptides could not form stable homodimers alone. Recombinant SS2 was phosphorylated by chloroplast protein kinases and recombinant casein kinase (CK) II at two N-terminal serine residues (S63, S65). Inhibition of chloroplast-dependent SS2 phosphorylation by heparin reinforced the role of stromal CK’s in this process. SS2 phosphorylation did not affect its catalytic activity, but monomeric SS2 was more active than homodimers, suggesting that SS2 activity might be regulated by its oligomeric conformation. Heteromeric enzyme complex (HEC) formation between SS2 and SBE2.2 was shown to be ATP-dependent, but not dependent on SS2 homodimerization or phosphorylation of SS2. Fluorophore-tagged SS2 was transiently expressed and localized in chloroplasts of tobacco (N. benthamiana). Stable transgenic lines of Arabidopsis thaliana were produced, in which ss2- plants were complemented with wild type and mutated SS2 sequences. In Arabidopsis, mRNAs for all SS2 constructs were detected in stable transgenic lines. Despite this, no recombinant SS2 protein was detected in the chloroplasts of any transformant. In summary, SS2 is post-translationally regulated by protein phosphorylation and homodimerization, for which its N-terminal region is required, and transitions between oligomeric states may regulate its catalytic activity. en_US
dc.language.iso en en_US
dc.rights Attribution-NonCommercial-NoDerivs 2.5 Canada *
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/2.5/ca/ *
dc.subject Starch en_US
dc.subject Starch synthase en_US
dc.subject Post-translational regulation en_US
dc.subject Intrinsic disorder en_US
dc.subject Phosphorylation en_US
dc.subject Protein-protein interactions en_US
dc.subject Metabolism en_US
dc.subject Arabidopsis en_US
dc.title Post-translational regulation of Arabidopsis thaliana starch synthase 2 en_US
dc.type Thesis en_US
dc.degree.programme Molecular and Cellular Biology en_US
dc.degree.name Doctor of Philosophy en_US
dc.degree.department Department of Molecular and Cellular Biology en_US
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