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Published on February 8, 2008; 10.1104/pp.108.116244

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Received January 14, 2008
Accepted February 7, 2008

Analysis of Protein Complexes in Wheat Amyloplasts Reveals Functional Interactions among Starch Biosynthetic Enzymes

Ian J. Tetlow , Kim G. Beisel , Scott Cameron , Amina Makhmoudova , Fushan Liu , Nicole S. Bresolin , Robin Wait , Matthew K. Morell , and Michael J. Emes *

Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada; Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, 1 Aspenlea Road, Hammersmith, London, W6 8LH, United Kingdom; Division of Plant Industry, CSIRO, Canberra ACT, GPO Box 1600, Australia

* Corresponding author; email: memes{at}uoguelph.ca.

Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat 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 kDa, and that SBEII forms may also exist as homo-dimers. Analysis of cross-linked 260 kDa 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 {gamma}-32P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260 kDa 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 kDa SS/SBEII protein complexes are formed around 10-15 days 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.




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