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Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

^a Departments of Botany and Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
^b Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, London, W6 8LH, United Kingdom
^c Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
^d School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom
^e Dipartmento di Biologia Vegetale, University of Naples (Federico II), 80139, Naples, Italy
^f Division of Plant Industry, Commonwealth Scientific and Industrial Research Organisation, Canberra ACT, Australia

¹ To whom correspondence should be addressed. E-mail itetlow{at}uoguelph.ca; fax 519-767-1991.

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with -³²P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated ³²P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule–associated phosphoproteins after incubation of intact amyloplasts with -³²P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein–protein interactions in the control of starch anabolism and catabolism.

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