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 Ca2+, Mg2+ and Mn2+ 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.
Highlights
Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) break down nucleoside triand diphosphates (NTPs/NDPs) to nucleoside monophosphates (NMPs) and inorganic phosphate (Pi) [1]
The biochemical analysis of the protein sequence confirmed that AtAPY1 contains the five apyrase conserved regions” (ACRs) typical of E-NTPDases and a single transmembrane domain (TM) near the N-terminus (Fig. 1)
The green fluorescent protein (GFP)-tag proved very useful in extracting active AtAPY1 and genetic complementation experiments gave no indication that the tag changed the biological function of AtAPY1 [22]
Summary
Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) break down nucleoside triand diphosphates (NTPs/NDPs) to nucleoside monophosphates (NMPs) and inorganic phosphate (Pi) [1]. E-NTPDases can be stimulated by a variety of divalent ions. A more salient characteristic of E-NTPDases, is the presence of five conserved domains called “apyrase conserved regions” (ACRs) [2,3,4]. E-NTPDases occur predominantly in eukaryotes where they function extracellularly as well as within the cell. “Ecto” refers to the outside orientation of the catalytic domain facing the extracellular space or the lumen of an organelle [5]. It was proposed to reserve their historical name “apyrase” for intracellular E-NTPDases [5]. In the plant literature, the term “apyrases” is often used for extracellular E-NTPDases as well
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