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Title: Oligomerization, membrane association and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase

Author
item SOARES, J S M - State University Of Campinas
item GENTILE, A - State University Of Campinas
item SCOSATO, V - State University Of Campinas
item DA C. LIMA, A - State University Of Campinas
item KIYOTA, E - State University Of Campinas
item DOS SANTOS, M - State University Of Campinas
item PAITTONI, C - Ciudad University, Argentina
item Huber, Steven
item APARICIO, R - State University Of Campinas
item MENOSSI, M - State University Of Campinas

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/15/2014
Publication Date: 11/28/2014
Citation: Soares, J., Gentile, A., Scosato, V., Da C. Lima, A., Kiyota, E., Dos Santos, M.L., Paittoni, C.V., Huber, S.C., Aparicio, R., Menossi, M. 2014. Oligomerization, membrane association and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase. Journal of Biological Chemistry. 289:33364-33377.

Interpretive Summary: Accumulation of sucrose in the storage stalk of sugarcane is the major determinant of crop quality and value. However, much remains to be learned about the mechanisms that control sugar accumulation. The present study was conducted to examine the role of the enayme, UDP-glucose pyrophosphorylase (UGPase), which catalyzes a reversible reaction that can function in pathways associated with sucrose synthesis or utilization. Expressing of the gene encoding UGPase (ScUGPase-1) decreased during stalk maturation and the expression level did not differ between two cultivars that accumulated different amounts of sucrose, suggesting a primary role for ScUGPase-1 in sucrose utilization. In vivo, the protein was found in both the soluble and membrane fractions, and in the soluble phase existed as a mixture of monomers and dimers. The dimer interfact was located in the carboxy-terminus of the protein, which also contain a phosphorylated residue in vivo. Enzyme activity was reversibly modulated by oxidation and reduction. Thus, UGPase activity in vivo may be controlled in many ways, and identifying the components is an important first step in understanding the important process of sucrose accumulation.

Technical Abstract: Sugarcane is a C4 plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms involved in sucrose accumulation in sugarcane are not well understood and little is known with regard to factors that control the extent of sucrose storage in stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an important enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights on ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with sucrose content in the internodes during development and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation and the enzyme activity was found to be affected by redox modification. Preincubation with H2O2 strongly inhibited activity and could be reversed by DTT. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser419 site in the soluble and membrane fractions from leaves, but not from internodes. Small-Angle X-ray Scattering analysis indicated that the dimer interface is located at the C-terminal and provided the first structural model of the dimer of sugarcane UGPase in solution.