far the major compound. It is of very high molecular Unite ´Mixte de Recherche NЊ111 du Centre National weight (10 7 -10 9 Da) and harbors 5% of ␣-1,6 branches de la Recherche Scientifique (reviewed by Manners, 1989). Amylose is often referred Universite ´des Sciences et Technologies de Lille to as a smaller linear molecule (molecular weight of 10 5 -59655 Villeneuve D'Ascq Cedex 10 6 Da) with very few ␣-1,6 branches (less than 1%). Its France association with amylopectin inside the granule remains † Exseed Genetics to be determined. Amylopectin is sufficient to generate 1568 Food Science Building full size granules both in wild-type starch from photosyn-Iowa State University thesizing cells and in mutant starches devoid of amy-Ames, Iowa 50011-1061 lose. No mutants lacking selectively amylopectin have ‡ Department of Biochemistry and Biophysics ever been described in plants suggesting that an under-Iowa State University standing of amylopectin biosynthesis will be sufficient to Ames, Iowa 50011 explain the major features of starch granule biogenesis.

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In maize kernels, mutations in the gene sugaryl (sul) result in (1) increased sucrose concentration; (2) decreased concentration of amylopectin, the branched component of starch; and (3) accumulation of the highly branched glucopolysaccharide phytoglycogen. To investigate further the mechanisms of storage carbohydrate synthesis in maize, part of the sul gene locus and a cDNA copy of the sul transcript were characterized. Five new sul mutations were isolated in a Mutator background, and the mutant allele sul-R4582::Mul was isolated by transposon tagging. The identity of the cloned element as the sul gene locus was confirmed by the cosegregation of restriction fragment length polymorphisms in the same or nearby genomic intervals with three additional, independent sul mutations. Pedigree analysis was also used to confirm the identity of sul. A 2.8-kb mRNA that is homologous to the cloned gene was detected in maize kernels, and a 2.7-kb cDNA clone was isolated based on hybridization to the genomic DNA. Specific portions of the cDNA hybridized with multiple segments of the maize genome, suggesting that sul is part of a multigene family. The cDNA sequence specified a polypeptide of at least 742 amino acids, which is highly similar in amino acid sequence to bacterial enzymes that hydrolyze a-(l-%) glucosyl linkages of starch. Therefore, debranching of glucopolysaccharides is seemingly part of the normal process of starch biosynthesis, and the final degree of branch linkages in starch most likely arises from the combined actions of branching and debranching enzymes.

(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...