Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts      

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.An important question in plant physiology is the means by which glucan storage homopolymers are synthesized such that they are able to assemble into semicrystalline starch granules. The starch polymer amylopectin consists of a-(1/4)-linked Glc units in linear chains, and these are joined to each other by a-(1/6) branch linkages. A distinguishing feature of amylopectin is that the branch points are clustered relative to each other (Thompson, 2000). The functional properties of starch depend on this ordered structure, which allows crystallization of the linear glucan chains that extend from the branch clusters. Packing of insoluble Glc units provides plants with a stable and abundant energy source to maintain metabolic needs in the absence of light. Considering that crystallization draws metabolic equilibria toward carbohydrate accumulation, another important physiological question is how the flux of reduced carbon is regulated such that seeds and other storage tissues achieve the proper balance of starch compared with protein and lipids.Biosynthesis of crystalline starch is accomplished in large part by the coordinated activities of starch synthases (SSs) and starch branching enzymes (SBEs), together with starch debranching enzymes (DBEs; Ball and Morell, 2003). SSs catalyze linear chain elongation by addition of a Glc unit donated from the nucleotide sugar ADP-Glc (ADPGlc) to the nonreducing end of an acceptor chain. Branch linkages are formed by the action of SBEs, whi...

A review of starch‐branching enzymes and their role in amylopectin biosynthesis

“…Studies with wheat and maize amyloplasts have shown that all SBE isoforms can be phosphorylated (8,9) and that protein phosphorylation regulates the association of SBE and SS isoforms in multienzyme protein complexes (see below; refs. 9,68,69).…”

Functional Interactions between Starch Synthase III and Isoamylase-Type Starch-Debranching Enzyme in Maize Endosperm

“…SSIII contains a highly conserved N-terminal extension beyond the catalytic region, referred to the SSIII-homology domain (SSIIIHD; Gao et al, 1998;Li et al, 2003;Ral et al, 2004;Dian et al, 2005). This domain has been implicated in binding both to starch and to other proteins that are active in starch biosynthesis (Hennen-Bierwagen et al, 2009;Wayllace et al, 2010). Thus, beyond its catalytic role, SSIII may physically coordinate the functions of other starch biosynthetic enzymes and/ or regulate their catalytic activities.…”

The integration of activity in saline environments: problems and perspectives

“…In addition, there are membrane complexes such as clathrin (Batchelder and Yarar 2010) and multifunctional enzymes in primary metabolism (e.g. pyruvate dehydrogenase, Behal et al 1993;Perham 2000), the Calvin cycle (Wedel et al 1997), the phenylpropanoid pathway (Winkel 2004), glycolysis (Graham et al 2007), polyamine biosynthesis (Alcázar et al 2011) and starch synthesis (Hennen-Bierwagen et al 2009;Liu et al 2009). Although some of the complexes 'make sense' based on well-known biochemistry, others, such as the association of aldolase with the vacuolar ATPase, are less obvious (Lu et al 2004).…”