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Post-translational Regulation of Starch Synthase IIa, a Key Enzyme of Starch Biosynthesis in Maize Endosperm

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Title: Post-translational Regulation of Starch Synthase IIa, a Key Enzyme of Starch Biosynthesis in Maize Endosperm
Author: Mehrpouyan, Sahar
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Emes, Michael J.
Abstract: Starch is the most abundant storage carbohydrate in plants, providing up to 70% of human caloric intake and has many industrial applications. Starch biosynthesis involves the coordination of three major enzyme activities, starch synthases (SSs), starch branching enzymes (SBEs) and debranching enzymes (DBEs). In cereals, some starch biosynthetic enzymes function via formation of heteromeric enzyme complexes (HECs) linked to protein phosphorylation. Previous studies have shown that the isozyme SSIIa forms HECs with SBEIIb, SSI, and SSIII which are functionally important in maize and other cereals. In this study, biochemical, bioinformatic, molecular and proteomic analyses were performed to investigate the post-translational mechanisms which act on maize SSIIa. The results imply a significant role for protein phosphorylation and protein-protein interactions in its regulation. Non-denaturing zymograms and western blots were used to detect the activity of amyloplast stromal SSIIa. Several SSIIa, electrophoretically-distinct bands were observed, the mobilities of which were markedly altered by conditions that favoured protein phosphorylation or dephosphorylation. Following gel permeation chromatography of amyloplast lysates, pre-treated with either ATP or alkaline phosphatase, elution profiles of SSIIa, SBEIIb and SSI were all substantially altered and variously coincident. The results suggest a dual role for phosphorylation in promoting association and dissociation of SSIIa-containing HECs in the maize amyloplast stroma. Furthermore, ATP treatment enhanced the catalytic activity of both endogenous and recombinant SSIIa in the presence of amyloplast stromal protein kinases and SBEIIb. Bioinformatic approaches were employed to investigate protein phosphorylation, and potential domains for interaction of SSIIa with SBEIIb. Mass spectrometric approaches were unsuccessful. Site-directed mutagenesis (SDM) of SSIIa identified the region(s) of protein phosphorylation and protein-protein interaction. 48 SDMs and truncations of SSIIa were produced, demonstrating that the central domain of SSIIa interacts with maize SBEIIb but is not phosphorylated. Data suggest that there are multiple phosphorylation sites within both N- and C-terminal regions of SSIIa, and one particular tyrosine residue (Tyr165) was identified. It was not possible to distinguish among multiple phospho-amino acids elsewhere. Results are discussed in the context of post-translational mechanisms, their role in cereal starch biosynthesis, and the potential to improve yield and modify starch.
Date: 2019-02

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