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Metabolism and Enzymology

Identification of Factors Regulating the Phosphorylation Status of Sucrose-Phosphate Synthase in Vivo ¹

U.S. Department of Agriculture, Agricultural Research Service, North Carolina State University, Raleigh, North Carolina 27695-7631, Department of Crop Science, North Carolina State University, Raleigh, North Carolina 27695-7631, Department of Botany, Plant Physiology Program, North Carolina State University, Raleigh, North Carolina 27695-7631

The purpose of this study was to identify the factors that control sucrose-phosphate synthase (SPS)-kinase and SPS-protein phosphatase (SPS-PP) activity in situ, and thereby mediate the activation of SPS by light or mannose. Feeding mannose to excised spinach (Spinacia oleracea) leaves in darkness resulted in a general sequestration of cellular phosphate (as evidenced by accumulation of mannose-6-P and depletion of glucose-6-P [Glc-6-P] and fructose-6-P [Fru-6-P]) and a relatively slow activation of SPS (maximum activation achieved within 90 min). Supplying exogenous inorganic phosphate (Pi) with mannose reduced sequestration of cellular Pi (as evidenced by mannose-6-P accumulation without depletion of hexose-P) and substantially reduced mannose activation of SPS. Thus, depletion of cytoplasmic Pi may be required for SPS activation; accumulation of mannose-6-P alone is clearly not sufficient. It was verified that Glc-6-P, but not mannose-6-P, was an inhibitor of partially purified SPS-kinase, and that Pi was an inhibitor of partially purified SPS-PP. Total extractable activity of SPS-kinase did not vary diurnally, whereas a pronounced light activation of SPS-PP activity was observed. Pretreatment of leaves in the dark with cycloheximide blocked the light activation of SPS-PP (assayed in vitro) and dramatically reduced the rate of SPS activation in situ (in saturating light and carbon dioxide). We conclude that rapid activation of SPS by light involves reduction in cytosolic Pi, an inhibitor of SPS-PP, and light activation of SPS-PP, by a novel mechanism that may involve (directly or indirectly) a protein synthesis step. An increase in cytosolic Glc-6-P, an inhibitor of SPS-kinase, would also favor SPS activation. Thus, the signal transduction pathway mediating the light activation of SPS involves elements of "fine" and "coarse" control.

¹ Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. Funded in part by the U.S. Department of Energy (DE-AI05-91ER20031 to S.C.H.). H.W. was supported by a fellowship from the Deutsche Forschungsgemeinschaft.

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