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

摘要

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. nnnn--------------------------------------------------------------------------------nAn 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 -(14)-linked Glc units in linear chains, and these are joined to each other by -(16) 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. nBiosynthesis 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, which cleave a linear chain and transfer the released fragment to a C6 hydroxyl group of the same or a neighboring chain. DBEs hydrolyze branch linkages, and genetic evidence indicates that this function is necessary in order for plants to accumulate crystalline starch (James et al., 1995; Ball et al., 1996; Myers et al., 2000). Multiple classes of SBE, SS, and DBE are highly conserved in the plant kingdom (Ball and Morell, 2003; Li et al., 2003; Leterrier et al., 2008). nnRecent 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). Further studies in maize (Zea mays) and wheat utilized combinations of yeast two-hybrid assays, affinity purification with immobilized recombinant ligands, and immunoprecipitation to demonstrate a large number of pair-wise interactions involving SSI, SSIIa, SSIII, SBEIIa, and SBEIIb (Hennen-Bierwagen et al., 2008; Tetlow et al., 2008). 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). Essentially all of the SSIII in the amyloplast extracts is in C670, and the great majority of SSIIa is in C300, suggesting that the physiological functions of these proteins derive from these assembly states. Understanding the functions of the complexes will require detailed characterization of their constituents, including any other binding partners that may be present. nnAmong these enzymes, SSIII has been implicated from several observations as a regulator of starch biosynthesis, in addition to its enzymatic role. Mutations in the maize gene dull1 (du1), which codes for SSIII, eliminated SSIII enzyme activity, as expected, and in addition simultaneously caused a major reduction in the activity of SBEIIa (Boyer and Preiss, 1981). The du1– mutation of maize or the equivalent genetic defect in rice (Oryza sativa) resulted in elevated total SS activity in soluble endosperm extracts as a result of increased SSI activity (Singletary et al., 1997; Cao et al., 1999; Fujita et al., 2007). In Arabidopsis (Arabidopsis thaliana) leaves, a regulatory role was indicated by the observation that mutations eliminating SSIII caused an increased rate of starch biosynthesis (Zhang et al., 2005). The mechanism(s) by which SSIII influences the activities of other starch biosynthetic enzymes or the overall starch biosynthesis rate is unknown. Part of the explanation may be that physical association of SSIII with other enzymes provides for regulatory interactions. nnRegulatory functions of SSIII proteins may be provided by an evolutionarily conserved amino acid sequence region located adjacent to the catalytic domain responsible for SS enzyme activity. Members of the conserved SSI, SSII, and SSIII classes of starch synthase all contain an N-terminal extension relative to the conserved catalytic domain homologous to glycogen synthase. In the SSI and SSII classes, the N-terminal extension is conserved among monocots and dicots but not universally throughout the land plants or in unicellular green algae. SSIII, in contrast, contains a region of approximately 450 residues immediately upstream of the catalytic region, referred to here as the SSIII homology domain (SSIIIHD), that appears to have been fixed in evolution as far back as the emergence of land plants (Gao et al., 1998; Li et al., 2000; Dian et al., 2005). For example, SSIIIHD from the monocot maize and the unicellular green alga Chlamydomonas reinhardtii share 32% identity over 415 aligned residues, and SSIIIHD from maize and the dicot Arabidopsis are 56% identical over 458 aligned residues. In contrast, the SSI N-terminal domain from maize is 16% identical over 92 aligned residues with Arabidopsis SSI and 12% identical over 72 aligned residues with Chlamydomonas SSI. SSIIIHD in maize is involved in protein-protein interactions with SSI (Hennen-Bierwagen et al., 2008) and in addition possesses sequences that confer a glucan-binding function (Palopoli et al., 2006; Senoura et al., 2007; Valdez et al., 2008). The further N-terminal extension of maize SSIII beyond the SSIIIHD domain, which is not conserved, has also been implicated in binding to other starch biosynthetic enzymes by yeast two-hybrid data (Hennen-Bierwagen et al., 2008). nnThis study further characterized multisubunit complexes containing SSIII and SSIIa. Maize mutations are available that eliminate particular starch biosynthetic enzymes in vivo, and using these tools the interdependence of specific SSs and SBEs for assembly into high molecular mass complexes was assessed. The results indicated that SSIII, SSIIa, SBEIIa, and SBEIIb associate together in an enzyme complex of approximately 670 kD, as opposed to individual or pair-wise high molecular mass assemblies. Consistent with the genetic results, biochemical analyses of amyloplast extracts demonstrated stable associations of SSIII with SSIIa, SBEIIb, and SBEIIa. Proteomic analyses revealed the presence of other proteins in the starch biosynthetic enzyme complexes. Two of these were large and small subunits of ADP-Glc pyrophosphorylase (AGPase), which catalyzes formation of the substrate of SS. Other enzymes not known to be directly involved in starch synthesis also were detected, including pyruvate orthophosphate dikinase (PPDK) and Suc synthase (SUS). In the instances of PPDK and SUS, inclusion in high molecular mass assemblies required the presence of multiple starch biosynthetic enzymes, indicating that the components are likely to all be in the same complex. These data revealed that specific enzymes from apparently distinct metabolic pathways interact with starch biosynthetic enzymes, suggesting potential means of coordinating and regulating carbon metabolism during grain filling.