Starch Biosynthetic Enzymes from Developing Maize Endosperm Associate in Multisubunit Complexes

Abstract

(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 accumulation, activities of key enzyme and gene expression in starch synthesis of wheat endosperm with different starch contents

“…Nevertheless, it should be kept in mind that the transcript levels are not always related to their enzymatic activities or protein levels, considering post-transcriptional and posttranslational controls (Sun et al 2005). There are intricate functional interactions between components of the starch biosynthetic system during wheat endosperm, and the starch-biosynthesizing system in higher plants demands the concerted expression of many genes at any particular developmental stage of a particular tissue (Tetlow et al 2004;Ohdan et al 2005;Hennen-Bierwagen et al 2008). In addition to maintaining the activity levels of individual enzymes, several combinations of protein-protein interactions, such as those among the wheat proteins for SBEI, SBEIIb, and DBE might play an essential role in the metabolic regulation of starch biosynthesis.…”

“…All plant varieties were grown in the field at ShiHeZi University, Shihezi, China, in March, 2007-2008 growing season. Crop management was the same as the local high-yield cultivation.…”

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).…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…There is evidence that several starch biosynthetic enzymes depend on other biosynthetic components for their activity; for example, a mutation affecting SSIII reduces the activity of SBEIIa whilst also increasing the activity of SSI in cell extracts [125], indicating that starch biosynthesis is most likely to be the result of several enzymes acting in a coordinated manner. The physical and biochemical interactions between starch-synthesising enzymes must thus be taken into account in order to fully understand the process of starch biosynthesis.…”

“…According to the specificities of individual isozymes, this complex was proposed to contribute to the synthesis of short and intermediate amylopectin branches to form the crystalline lamellae [127,130]. In addition, maize SSI, SSIIa, SSIII, SBEI, SBEIIa and SBEIIb (SSIIa, SSIII, SBEIIa, SBEIIb in ~600 kDa complex; SSIIa, SBEIIa, SBEIIb in ~300kDa complex) were found within protein-protein complexes [125]. A larger protein complex including SSIIa, SSIIIa, SSIVb, SBEI, SBEIIb and PUL (>700 kDa) was eluted by gel filtration chromatography from soluble rice endosperm extracts [131].…”