Plant Physiology 134:1427-1438 (2004)
© 2004 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
Phosphorylation of the Amino Terminus of Maize Sucrose Synthase in Relation to Membrane Association and Enzyme Activity1
Shane C. Hardin,
Heike Winter and
Steven C. Huber*
United States Department of Agriculture, Agricultural Research Service, Photosynthesis Research Unit, and Departments of Plant Biology and Crop Sciences, University of Illinois, Urbana, Illinois 61801 (S.C.H., S.C.H.); and Department of Botany, North Carolina State University, Raleigh, North Carolina 27695-7247 (H.K.)
Sucrose synthase (SUS) is phosphorylated on a major, amino-terminal site located at Ser-15 (S15) in the maize (Zea mays) SUS1 protein. Site- and phospho-specific antibodies against a phosphorylated S15 (pS15) peptide allowed direct analysis of S15 phosphorylation in relation to membrane association. Immunoblots of the maize leaf elongation zone, divided into 4-cm segments, demonstrated that the abundance of soluble (s-SUS) and membrane (m-SUS) SUS protein showed distinct positional profiles. The content of m-SUS was maximal in the 4- to 8-cm segment where it represented 9% of total SUS and occurred as a peripheral membrane protein. In contrast, s-SUS was highest in the 12- to 16-cm segment. Relative to s-SUS, m-SUS was hypophosphorylated at S15 in the basal 4 cm but hyperphosphorylated in apical segments. Differing capabilities of the anti-pS15 and anti-S15 peptide antibodies to immunoprecipitate SUS suggested that phosphorylation of S15, or exposure of unphosphorylated SUS to slightly acidic pH, altered the structure of the amino terminus. These structural changes were generally coincident with the increased sucrose cleavage activity that occurs at pH values below 7.5. In vitro S15 phosphorylation of the S170A SUS protein by a maize calcium-dependent protein kinase (CDPK) significantly increased sucrose cleavage activity at low pH. Collectively, the results suggest that (1) SUS membrane binding is controlled in vivo; (2) relative pS15 content of m-SUS depends on the developmental state of the organ; and (3) phosphorylation of S15 affects amino-terminal conformation in a way that may stimulate the catalytic activity of SUS and influence membrane association.
1 This research was supported in part by funds from the U.S. Department of Energy (grant no. DE-AI05-91ER20031 to S.C.H.).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036780.
* Corresponding author; e-mail schuber1{at}life.uiuc.edu; fax 217-244-4419.
Received November 25, 2003;
returned for revision January 6, 2004;
accepted January 6, 2004.
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