title: The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.
abstract: The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+), Mg(2+) and Mn(2+) were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling.
authors.affiliation: Department of Biology, Technische Universität Dresden, Dresden, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Massalski
authors.affiliation: Department of Biology, Technische Universität Dresden, Dresden, Germany.
authors.forename: Jeannine
authors.initials: J
authors.lastname: Bloch
authors.affiliation: Division of Structural Biology, University of Oxford, Oxford, United Kingdom.
authors.forename: Matthias
authors.initials: M
authors.lastname: Zebisch
authors.affiliation: Department of Biology, Technische Universität Dresden, Dresden, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Steinebrunner
mesh_terms: D000595:Amino Acid Sequence; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D054730:Protein Interaction Domains and Motifs; D011687:Purines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; D011687:Purines; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; C522733:APY1 protein, Arabidopsis; D001081:Apyrase
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