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First published online February 8, 2008; 10.1104/pp.108.116244 Plant Physiology 146:1878-1891 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Analysis of Protein Complexes in Wheat Amyloplasts Reveals Functional Interactions among Starch Biosynthetic Enzymes1,[C],[W],[OA]Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada (I.J.T., K.G.B., S.C., A.M., F.L., N.S.B., M.J.E.); Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, London W6 8LH, United Kingdom (R.W.); and Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra, Australian Capital Territory 2601, Australia (M.K.M.)
Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with
1 This work was supported by the Natural Sciences and Engineering Research Council Grant (no. 262209 to M.J.E.), the Ontario Ministry of Agriculture and Food Bio-Products Research Grant (project no. 026262 to I.J.T. and M.J.E.), and the New South Wales Agricultural Genomics Centre (to M.K.M.). 2 Present address: Technische Universität Kaiserslauten, Abteilung Pflanzenphysiologie, Erwin-Schrödinger-Strasse 22, D–67633, Kaiserslautern, Germany. 3 Present address: Centre for Research in Neuroscience, University of Montreal, Sherbrooke Street West, Montreal, Quebec, H3A 2T5, Canada. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Michael J. Emes (memes{at}uoguelph.ca). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116244 * Corresponding author; e-mail memes{at}uoguelph.ca. Received January 14, 2008; accepted February 7, 2008; published February 8, 2008. Related articles in Plant Physiol.:
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-32P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis. 
