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ARS Home » Research » Publications at this Location » Publication #168757


item Hardin, Shane
item Huber, Steven

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/6/2004
Publication Date: 4/1/2004
Citation: Hardin, S.C., Winter, H., Huber, S.C. 2004. Phosphorylation of the amino-terminus of maize sucrose synthase in relation to membrane association and enzyme activity. Plant Physiology. 134:1427-1438.

Interpretive Summary: Sucrose synthase is an important enzyme of sucrose metabolism in growing plant organs, such as developing seeds, and channels carbon from sucrose into various metabolic and biosynthetic pathways. The activity of sucrose synthase in a growing organ is thought to be a marker for, or possible determinant of, growth. Consequently, there is great interest in identifying factors that control the activity and function of this critical plant enzyme. Sucrose synthase has been known to be modified by a mechanism, known as protein phosphorylation that involves covalent attachment of a phosphate molecule to a specific amino acid. In the case of sucrose synthase, phosphate is attached to a specific serine residue near the amino terminus of the protein. While phosphorylation has been known to occur, the effects of phosphorylation on enzyme parameters is still not clear. In the present study, specific antibodies were used to show that phosphorylation affects the conformation of the amino terminus of the protein, most likely by favoring an open-coil conformation rather than an alpha-helix. This conformational change may be responsible for an increase in enzyme activity and changes in binding to membranes that are observed. Elucidating the basis for control of plant metabolism by protein phosphorylation may provide rationale approaches to control specific aspects of metabolism. These results add substantially to our understanding of the control of sucrose metabolism by protein phosphorylation and thus will be of interest to a wide audience of plant scientists. Long-standing controversies concerning the localization of the sucrose synthase protein and the general role of phosphorylation are resolved using modification-specific antibodies. The results lay the foundation for future attempts to control this important enzyme using molecular genetic manipulation.

Technical Abstract: Sucrose synthase (SUS) is phosphorylated on a major, amino-terminal site located at serine 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 hypo-phosphorylated at S15 in the basal 4-cm but hyper-phosphorylated in apical segments. Differing capabilities of the anti-pS15 and anti-S15 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: i) SUS membrane binding is controlled in vivo; ii) relative pS15 content of m-SUS depends on the developmental state of the organ; and iii) phosphorylation of S15 affects amino-terminal conformation in a way that may stimulate the catalytic activity of SUS and influence membrane association.