title: Oligomerization, membrane association, and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase.
abstract: Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are 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 the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the 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 intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.
authors.affiliation: From the Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, C.P. 6109, Campinas, SP, Brazil.
authors.forename: Jose Sergio M
authors.initials: JS
authors.lastname: Soares
authors.affiliation: From the Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, C.P. 6109, Campinas, SP, Brazil.
authors.forename: Agustina
authors.initials: A
authors.lastname: Gentile
authors.affiliation: the Laboratório de Biologia Estrutural e Cristalografia, Instituto de Química, Universidade Estadual de Campinas, C.P. 6154, Campinas, SP, Brazil.
authors.forename: Valeria
authors.initials: V
authors.lastname: Scorsato
authors.affiliation: the Laboratório de Biologia Estrutural e Cristalografia, Instituto de Química, Universidade Estadual de Campinas, C.P. 6154, Campinas, SP, Brazil.
authors.forename: Aline da C
authors.initials: Ada C
authors.lastname: Lima
authors.affiliation: the Laboratório de Biologia Estrutural e Cristalografia, Instituto de Química, Universidade Estadual de Campinas, C.P. 6154, Campinas, SP, Brazil.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Kiyota
authors.affiliation: the Centro de Ciências Exatas e Tecnologia, Núcleo de Química, Universidade Federal do Sergipe, C.P. 49500000, Itabaiana, SE, Brazil.
authors.forename: Marcelo Leite
authors.initials: ML
authors.lastname: Dos Santos
authors.affiliation: the Instituto de Agrobiotecnologia del Litoral (UNL-CONICET), Universidad Nacional del Litoral, Ciudad Universitaria-Paraje El Pozo, CC242, S3000ZAA Santa Fe, Argentina.
authors.forename: Claudia V
authors.initials: CV
authors.lastname: Piattoni
authors.affiliation: the Department of Agriculture Agricultural Research Service, and Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Huber
authors.affiliation: the Laboratório de Biologia Estrutural e Cristalografia, Instituto de Química, Universidade Estadual de Campinas, C.P. 6154, Campinas, SP, Brazil.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Aparicio
authors.affiliation: From the Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, C.P. 6109, Campinas, SP, Brazil, menossi@lgf.ib.unicamp.br.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Menossi
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D008958:Models, Molecular; D010766:Phosphorylation / Q000502:physiology; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D018547:Plant Stems / Q000737:chemistry / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D031786:Saccharum / Q000201:enzymology*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000096:biosynthesis* / Q000737:chemistry; D014532:Uridine Diphosphate Glucose / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D010940:Plant Proteins; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose
keywords: Gene Expression; Kinetics; Protein Phosphorylation; Redox Regulation; Small Angle X-ray Scattering (SAXS); Sucrose