Abstract
Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.
Highlights
Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; the nature of those assemblies remains to be defined
Suc synthase (SUS)-SH1, ADP-Glc pyrophosphorylase (AGPase), and pyruvate orthophosphate dikinase (PPDK) have been highlighted here because they are found associated with starch biosynthetic enzymes by multiple methods
Other proteins identified in the proteomic studies only from the copurification analyses are likely to be amyloplast proteins that happen to coelute with the starch biosynthetic enzyme complexes without being part of the same quaternary structure, such as the many heat shock proteins present
Summary
Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Recent evidence indicates that certain SSs and SBEs are capable of physically associating with each other (Tetlow et al, 2004a, 2004b, 2008; Hennen-Bierwagen et al, 2008) The first such evidence came from analysis of amyloplast extracts from developing wheat (Triticum aestivum) endosperm, showing that SBEI, SBEIIb, and starch phosphorylase coimmunoprecipitate and that phosphorylation of one or more of those proteins is necessary for the association (Tetlow et al, 2004b). Gel permeation chromatography (GPC) analyses of maize amyloplast extracts demonstrated the existence of complexes in elution peaks corresponding to approximately 670 and 300 kD (referred to as C670 and C300, respectively) that contained SSIII, SSIIa, SBEIIa, and SBEIIb in varying relative concentrations (Hennen-Bierwagen et al, 2008). Understanding the functions of the complexes will require detailed characterization of their constituents, including any other binding partners that may be present
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