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Regulation of Maize (Zea mays L.) Starch Synthase IIa by Protein Phosphorylation

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Title: Regulation of Maize (Zea mays L.) Starch Synthase IIa by Protein Phosphorylation
Author: Rayirath, Usha
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Emes, Michael
Abstract: Starch is a significant carbohydrate reserve in plants and has enormous use in both food and non-food industries. Biosynthesis of storage starch in maize (Zea mays L.) occurs in the amyloplasts of the developing endosperm, through the coordinated actions of several enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE) and debranching enzymes (DBE). Starch synthase IIa (SSIIa) catalyzes the synthesis of intermediate glucan chains (DP 12-25) and plays a significant role in starch biosynthesis. Previous studies indicate that in cereal endosperm, protein phosphorylation plays a major role in regulating the formation of functional multi-enzyme complexes between SSs and SBEs during starch biosynthesis and that SSIIa forms the core of such a functional protein complex, with SSI and SBEIIb. The present study investigated the specific role of protein phosphorylation on the regulation of SSIIa, in the amyloplasts of developing maize endosperm, during starch biosynthesis. In vitro phosphorylation of stromal SSIIa in maize amyloplasts was detected by phosphate affinity Mn2+ Phos-tagTM gel electrophoresis, Pro-Q® Diamond phospho-protein gel staining, and by autoradiography following radio labelling with γ-[32-P] ATP. The results indicated that granule bound SSIIa exists in the phosphorylated state. In vitro phosphorylation of recombinant maize SSIIa and immunopurified SSIIa occurred only in the presence of amyloplast lysates and could be inhibited by protein kinase inhibitors, suggesting the presence of one or more protein kinase(s) in amyloplasts. ATP caused marked shifts in the electrophoretic mobility of SSIIa in non-denaturing polyacrylamide gels, and also in phosphate affinity (Phos-tag) gels, further suggesting the role of post-translational protein phosphorylation in regulating maize SSIIa. Protein phosphorylation significantly enhanced (12-fold), and dephosphorylation substantially reduced the catalytic activity (Vmax) of maize SSIIa, whereas its dissociation constant (Kd) and affinity for amylopectin was not affected. Depending on the phosphorylation status, stromal maize SSIIa existed in two distinct protein complexes, a LMW (260 kDa) protein complex was formed with SSI and SBEIIb under conditions favouring phosphorylation; whereas under conditions favouring dephosphorylation, this 260 kDa complex of SSIIa-SSI-SBEIIb possibly associated with other starch synthesizing enzymes and/or itself to form HMW complexes of 670 kDa or more.
URI: http://hdl.handle.net/10214/8076
Date: 2014-05
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