title: Multimeric states of starch phosphorylase determine protein-protein interactions with starch biosynthetic enzymes in amyloplasts.
abstract: Protein-protein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucan-synthetic and phosphorolytic directions. Protein-protein interaction experiments employing the recombinant SP as an affinity ligand with amyloplast extracts showed that the multimeric state of SP determined interactions with other enzymes of the starch biosynthetic pathway. The monomeric form of SP interacts with starch branching enzyme I (SBEI) and SBEIIb, whereas only SBEI interacts with the tetrameric form of SP. In all cases, protein-protein interactions were broken when amyloplast lysates were dephosphorylated in vitro, and enhanced following pre-treatment with ATP, suggesting a mechanism of protein complex formation regulated by protein phosphorylation. In vitro protein phosphorylation experiments with [γ-(32)P]-ATP show that SP is phosphorylated by a plastidial protein kinase. Evidence is presented which suggests SBEIIb modulates the catalytic activity of SP through the formation of a heteromeric protein complex.
authors.affiliation: Department of Molecular and Cellular Biology, Science Complex, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Renuka M
authors.initials: RM
authors.lastname: Subasinghe
authors.affiliation: Department of Molecular and Cellular Biology, Science Complex, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Fushan
authors.initials: F
authors.lastname: Liu
authors.affiliation: Department of Molecular and Cellular Biology, Science Complex, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Ursula C
authors.initials: UC
authors.lastname: Polack
authors.affiliation: Department of Plant Agriculture, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Lee
authors.affiliation: Department of Molecular and Cellular Biology, Science Complex, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Emes
authors.affiliation: Department of Molecular and Cellular Biology, Science Complex, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Electronic address: itetlow@uoguelph.ca.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Tetlow
mesh_terms: D010766:Phosphorylation; D018087:Plastids / Q000378:metabolism*; D011485:Protein Binding; D013213:Starch / Q000096:biosynthesis*; D024941:Starch Phosphorylase / Q000378:metabolism*
chemical_list: D013213:Starch; D024941:Starch Phosphorylase
keywords: Amylopectin; Amyloplast; Protein phosphorylation; Protein–protein interactions; Starch branching enzyme; Starch phosphorylase; Starch synthesis