Characterization of <i>ADG1</i>, an <i>Arabidopsis</i> locus encoding for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability

Wang, Shue-Mei; Lue, Wei-Ling; Yu, Tien‐Shin; Long, Jih-hau; Wang, Chen-nai; Eimert, Klaus; Chen, Jychian

doi:10.1046/j.1365-313x.1998.00009.x

Cited by 58 publications

(69 citation statements)

References 36 publications

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“…The OtaL protein could not be expressed in a soluble form, possibly because it needs the S subunit for folding and solubility as demonstrated in other organisms (4,11,37). When OtaL was expressed alone, it went to the insoluble fraction after ultrasonic disruption of the cells.…”

Section: Resultsmentioning

confidence: 99%

“…They have good expression by themselves but have defective regulatory properties (low apparent affinity for the activator 3-PGA) (1, 2). OtaL, StuL, and Arabidopsis APL3 and APL4 isoforms confer a high apparent affinity for the activator to the heterotetramer (1, 2) but cannot fold properly by themselves (2,11,37). There are two remarkable differences between O. tauri S and L subunits and other ADP-Glc PPases.…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, the presence of two types of subunits may have constrained each subunit to adopt complementary roles. For instance, S and L subunits need to interact to form tetramers in vivo (11). Because fewer S isoforms (e.g.…”

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confidence: 99%

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Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Kuhn¹,

Falaschetti²,

Ballícora

2009

Journal of Biological Chemistry

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ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (V max /K m ) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths.Starch synthesis in photosynthetic eukaryotes such as higher plants and unicellular algae is controlled by a heterotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase, 4 EC 2.7.7.27). This enzyme catalyzes the reaction of ATP and Glc-1-P to form ADP-glucose (ADP-Glc) and PP i , and it is primarily activated by 3-phosphoglycerate (3-PGA) and inhibited by P i . In photosynthetic eukaryotes the enzyme includes two distinct S and L homologous subunits (S 2 L 2 or ␣ 2 ␤ 2 ), but in photosynthetic bacteria the enzyme is a homotetramer (␣ 4 ). There has been debate regarding the role of the different subunits of ADP-Glc PPase. The S subunit homotetramer from potato (Solanum tuberosum) tuber (StuS), Arabidopsis thaliana (APS1), and barley (Hordeum vulgare) endosperm have a catalytic function with defective regulatory properties (1-3). It is not clear if there is a set of universal roles for the L subunit in plants. The L subunit from potato tuber (StuL) is catalytically deficient and plays more of a regulatory role by modifying the apparent affinity of the S subunit toward allosteric regulators (1, 4). On the other hand, the Arabidopsis APL1 and APL2 isoforms have both catalytic and regulatory functions, whereas the APL3 and APL4 isoforms behave like StuL (2, 5). The maize (Zea mays) endosperm L subunit...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Kuhn¹,

Falaschetti²,

Ballícora

2009

Journal of Biological Chemistry

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“…To rule out the possibility that the low rate of oil accumulation in sse1 was simply a consequence of increased carbon flux into starch, we assessed the effect of abolishing starch synthesis on the production of oil in sse1. As shown in Figure 5B, although starch accumulation was prevented by introducing the starch synthesis mutants adg1 (Lin et al, 1988;Wang et al, 1998) or pgm1 (Caspar et al, 1985, Periappuram et al, 2000 into the sse1 background, the levels of oil in the sse1adg1 or sse1pgm1 double mutants and the sse1 single mutant (all in the C24 and Col hybrid background) were indistinguishably low, less than 10% of that in wild type C24 or Col. These results demonstrated that the low rate of oil deposition in sse1 was not caused by the increased starch synthesis.…”

Section: Sse1 Developing Embryos Exhibit Reduced Rates Of Fatty Acid mentioning

confidence: 97%

The Peroxisome Deficient Arabidopsis Mutant sse1 Exhibits Impaired Fatty Acid Synthesis

2004

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The Arabidopsis Shrunken Seed 1 (SSE1) gene encodes a homolog of the peroxisome biogenesis factor Pex16p, and a loss-of-function mutation in this gene alters seed storage composition. Two lines of evidence support a function for SSE1 in peroxisome biogenesis: the peroxisomal localization of a green fluorescent protein-SSE1 fusion protein and the lack of normal peroxisomes in sse1 mutant embryos. The green fluorescent protein-SSE1 colocalizes with the red fluorescent protein (RFP)-labeled peroxisomal markers RFP-peroxisome targeting signal 1 and peroxisome targeting signal 2-RFP in transgenic Arabidopsis. Each peroxisomal marker exhibits a normal punctate peroxisomal distribution in the wild type but not the sse1 mutant embryos. Further studies reported here were designed toward understanding carbon metabolism in the sse1 mutant. A time course study of dissected embryos revealed a dramatic rate decrease in oil accumulation and an increase in starch accumulation. Introduction of starch synthesis mutations into the sse1 background did not restore oil biosynthesis. This finding demonstrated that reduction in oil content in sse1 is not caused by increased carbon flow to starch. To identify the blocked steps in the sse1 oil deposition pathway, developing sse1 seeds were supplied radiolabeled oil synthesis precursors. The ability of sse1 to incorporate oleic acid, but not pyruvate or acetate, into triacylglycerol indicated a defect in the fatty acid biosynthetic pathway in this mutant. Taken together, the results point to a possible role for peroxisomes in the net synthesis of fatty acids in addition to their established function in lipid catabolism. Other possible interpretations of the results are discussed.

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“…The higher plant SS group can be divided in two subgroups: one corresponding to monocots and a second one corresponding to dicots (including APS1 from Arabidopsis). APS1 from Arabidopsis is essential to starch synthesis in vivo as revealed the study of starch-lacking mutant plants (45). Small subunits are considered to be ubiquitous, although in the case of rice, Vicia faba, and pea, for which two genes coding for …”

Section: Kinetic Parameters Of the Arabidopsis Recombinant Adp-glc Ppmentioning

confidence: 99%

The Different Large Subunit Isoforms of Arabidopsis thaliana ADP-glucose Pyrophosphorylase Confer Distinct Kinetic and Regulatory Properties to the Heterotetrameric Enzyme

Crevillén

Ballícora

Mérida

et al. 2003

Journal of Biological Chemistry

125

153

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ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according to the necessities for starch synthesis in a given tissue.

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Characterization of ADG1, an Arabidopsis locus encoding for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability

Cited by 58 publications

References 36 publications

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

The Peroxisome Deficient Arabidopsis Mutant sse1 Exhibits Impaired Fatty Acid Synthesis

The Different Large Subunit Isoforms of Arabidopsis thaliana ADP-glucose Pyrophosphorylase Confer Distinct Kinetic and Regulatory Properties to the Heterotetrameric Enzyme

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