(F.L., I.J.T., M.J.E.)Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system, including starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes; however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or starch debranching enzymes associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between three separate SS polypeptides (SSI, SSIIa, and SSIII) and three separate BE polypeptides (BEI, BEIIa, and BEIIb) from maize (Zea mays) amyloplasts. Detection methods included in vivo protein-protein interaction tests in yeast (Saccharomyces cerevisiae) nuclei, immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand. Eight different instances were detected of specific pairs of proteins associating either directly or indirectly in the same multisubunit complex, and direct, pairwise interactions were indicated by the in vivo test in yeast. In addition, SSIIa, SSIII, BEIIa, and BEIIb all comigrated in gel permeation chromatography in a high molecular mass form of approximately 600 kD, and SSIIa, BEIIa, and BEIIb also migrated in a second high molecular form, lacking SSIII, of approximately 300 kD. Monomer forms of all four proteins were also detected by gel permeation chromatography. The 600-and 300-kD complexes were stable at high salt concentration, suggesting that hydrophobic effects are involved in the association between subunits.Plant species typically store reduced carbon in the glucan polymer amylopectin, located in semicrystalline, insoluble starch granules. Amylopectin has the same chemical nature as glycogen, the soluble glucan storage polymer present in most nonplant species. Glc residues in both polymers are linked in linear chains by a-(1/4) glycoside bonds, and these are joined by a-(1/6) glycoside bonds referred to as branch linkages. Amylopectin and glycogen differ in molecular architecture, however, with regard to branch frequency and the relative positions of the a-(1/6) bonds. Branch linkages of amylopectin are located in clusters relatively close to one another compared to the longer interbranch distances of glycogen (Thompson, 2000). Also, the branch frequency is lower in amylopectin than glycogen. These architectural features likely allow amylopectin crystallization and thus explain the different physical properties of starch and glycogen.The chemical structures of amylopectin and glycogen are produced by the same classes of enzyme, specifically a glucan synthase that transfers Glc residues to a growing linear chain from a nucleotide sugar donor and a glucan branching enzyme that cleaves a linear chain at a glycoside bond and transfers one portion of it to a C-6 hydroxyl. A possible ex...

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...

Functions of isoamylase-type starch-debranching enzyme (ISA) proteins and complexes in maize (Zea mays) endosperm were characterized. Wild-type endosperm contained three high molecular mass ISA complexes resolved by gel permeation chromatography and native-polyacrylamide gel electrophoresis. Two complexes of approximately 400 kD contained both ISA1 and ISA2, and an approximately 300-kD complex contained ISA1 but not ISA2. Novel mutations of sugary1 (su1) and isa2, coding for ISA1 and ISA2, respectively, were used to develop one maize line with ISA1 homomer but lacking heteromeric ISA and a second line with one form of ISA1/ISA2 heteromer but no homomeric enzyme. The mutations were su1-P, which caused an amino acid substitution in ISA1, and isa2-339, which was caused by transposon insertion and conditioned loss of ISA2. In agreement with the protein compositions, all three ISA complexes were missing in an ISA1-null line, whereas only the two higher molecular mass forms were absent in the ISA2-null line. Both su1-P and isa2-339 conditioned near-normal starch characteristics, in contrast to ISA-null lines, indicating that either homomeric or heteromeric ISA is competent for starch biosynthesis. The homomer-only line had smaller, more numerous granules. Thus, a function of heteromeric ISA not compensated for by homomeric enzyme affects granule initiation or growth, which may explain evolutionary selection for ISA2. ISA1 was required for the accumulation of ISA2, which is regulated posttranscriptionally. Quantitative polymerase chain reaction showed that the ISA1 transcript level was elevated in tissues where starch is synthesized and low during starch degradation, whereas ISA2 transcript was relatively abundant during periods of either starch biosynthesis or catabolism.Starch biosynthesis is a central function in plant metabolism that is accomplished by a multiplicity of conserved enzymatic activities. Two known activities are starch synthase, which catalyzes the polymerization of glucosyl units into a(1/4)-linked "linear" chains, and starch-branching enzyme, which catalyzes the formation of a(1/6) glycoside bond branches that join linear chains. Acting together, the starch synthases and starch-branching enzymes assemble the relatively highly branched polymer amylopectin, with approximately 5% of the glucosyl residues participating in a (1/6) bonds, and the lightly branched molecule amylose. Amylopectin and amylose assemble into semicrystalline starch granules, which in land plants and green algae are located in plastids.A third activity necessary for normal starch biosynthesis is provided by starch-debranching enzyme (DBE), which hydrolyzes a(1/6) linkages. Two DBE classes have been conserved separately in plants (Beatty et al., 1999). These are referred to here as pullulanase-type DBE (PUL) and isoamylase-type DBE (ISA), based on similarity to prokaryotic enzymes with particular substrate specificity. ISA function in starch production is implied from genetic observations that mutations typically result in reduced ...