The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space

Schiller et al. BMC Plant Biology 0 DFG-Center for Regenerative Therapies Dresden (CRTD), Technische Universitat Dresden , Fetscherstrae 105, Dresden 01307 , Germany 1 Department of Biology, Section of Molecular Biotechnology, Technische Universitat Dresden , Helmholtzstrae 10, Dresden 01069 , Germany - The Arabidopsis apyrase the Golgi instead of the AtAPY1 is localized in extracellular space Schiller et al. Open Access The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space Madlen Schiller1, Carolin Massalski1, Thomas Kurth2 and Iris Steinebrunner1* Background: The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function. Results: Transgenic Arabidopsis lines expressing AtAPY1 fused to the SNAP-(O6-alkylguanine-DNA alkyltransferase)-tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing AtAPY1-GFP by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, AtAPY1-GFP expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide) and crossed with a transgenic line expressing the trans-Golgi marker Rab E1d. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing AtAPY1-GFP and either the fluorescent protein-tagged Golgi marker Membrin 12, Syntaxin of plants 32 or Golgi transport 1 protein homolog showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea (not alkaline or high salt conditions) were able to release the AtAPY1 protein from microsomal membranes. Conclusions: AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is therefore not likely to regulate extracellular nucleotide signals as previously thought. We propose instead that AtAPY1 exerts its growth and developmental effects by possibly regulating glycosylation reactions in the Golgi. Background The term apyrase (adenosine pyrophosphatase) for an enzyme cleaving the phosphoanhydride bonds of ATP and ADP was coined by Otto Meyerhof in 1945 [1]. Decades later, the alternative name NTPDase (nucleoside triphosphate diphosphohydrolase) was officially proposed [2] because apyrases hydrolyze a wide range of nucleoside triand diphosphates (reviewed in [3]). Apyrases have been found in many pro- and eukaryotes (reviewed in [3]), and they all share highly conserved regions [4]. In plants, the * Correspondence: 1Department of Biology, Section of Molecular Biotechnology, Technische Universitt Dresden, Helmholtzstrae 10, Dresden 01069, Germany Full list of author information is available at the end of the article postulated functions are diverse and include nodulation [5-9], resistance to xenobiotics [10], phosphate scavenging [11] and growth [12-16]. Each eukaryotic genome screened for the presence of apyrase genes holds at least two candidates. In Arabidopsis thaliana, a total of seven apyrase gene candidates exist. Our research focused on the function of the two Arabidopsis apyrase genes AtAPY1 and AtAPY2, whose corresponding proteins share an identity of 87% amino acids. Knocking out one of the two apyrase genes by T-DNA (transfer DNA) insertion resulting in an apy1 or apy2 single knockout (SKO) caused no obvious differences in phenotype compared with the wild type (WT) [17], but knocking out both AtAPY1 and AtAPY2 inhibited pollen germination [17] and was seedling-lethal [18]. Overexpression of either AtAPY1 or AtAPY2 led to more vigorous growth of hypocotyls and pollen tubes [12]. Suppression of expression, however, by RNA interference targeting AtAPY1 in the apy2 SKO background, inhibited growth throughout the whole plant and especially in the hypocotyls and roots [12]. Several lines of evidence suggested that these growth effects are mediated by AtAPY1 and AtAPY2 regulating extracellular ATP (eATP) signals [12]: Apyrase activity, measured in the extracellular matrix (ECM) of growing pollen tubes, could be reduced by adding chemical inhibitors or polyclonal antibodies directed against AtAPY1. The reduction in activity simultaneously raised eATP levels and reduced pollen tube growth [12]. These findings explained the inhibition of growth when the expression of AtAPY1 and AtAPY2 is suppressed or shut off and provided the first direct evidence that apyrases function as regulators of extracellular nucleotides such as eATP in plants. In the animal field, the direct link between ecto-apyrases and [eATP] had already been shown [19]. Similarly, eATP was already known to serve as signaling molecule in animals (reviewed in [20]) before it became recognized as such in plants in the past decade (reviewed in [21-23]). The objective of this study was to confirm the extracellular function of the two Arabidopsis apyrases AtAPY1 and AtAPY2 by their localization to the plasma membrane or the apoplast. Since AtAPY1 and AtAPY2 were shown to be functionally redundant in their ability to rescue pollen germination of double knockout apyrase (DKO) pollen [17] and seedling viability in DKO mutants [18], an overlapping subcellular localization of the two apyrases was likely. Therefore, this study focused on the localization of only one apyrase. Stable Arabidopsis lines were generated expressing AtAPY1 fused to either one of two tag sequences, SNAP or GFP. For the identification of the AtAPY1-positive compartments, organelle-specific marker proteins were co-expressed and immunogold labeling was used. Unexpectedly, the apyrase was not localized to the plasma membrane or cell wall, but to the Golgi apparatus. Methods Plant material and growth conditions For all experiments, the A. thaliana ecotype Wassilewskija was used as the WT control. Seedlings were grown for one week under sterile conditions on agar plates (4.3 gL-1Murashige Skoog (MS) salts, 0.5 g L-1 MES, pH 5.7 (adjusted with KOH), 1% (w/v) sucrose, 0.8% (w/v) agar) or in liquid medium (see above, without agar) under shaking (80 rpm). After one week on agar plates, seedlings were transferred to soil (Einheitserde, type P, Ptzer Inc., Sinntal-Jossa, Germany) and grown at 24C (...truncated)