Biochimica et Biophysica Acta 1850 (2015) 1921–1929

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High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana Christopher Girke a, Elena Arutyunova b, Maria Syed b, Michaela Traub a, Torsten Möhlmann a, M. Joanne Lemieux b,⁎ a b

Department of Plant Physiology, University of Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern, Germany Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

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Article history: Received 12 January 2015 Received in revised form 31 May 2015 Accepted 11 June 2015 Available online 12 June 2015 Keywords: Equilibrative nucleoside transporter Arabidopsis thaliana ENT7 Nucleoside Nucleobase Membrane transport Recombinant expression Reconstitution Microscale thermophoresis MST Fluorescence size exclusion chromatography FSEC

a b s t r a c t Background: Equilibrative nucleoside transporters (ENTs) facilitate the import of nucleosides and their analogs into cells in a bidirectional, non-concentrative manner. However, in contrast to their name, most characterized plant ENTs act in a concentrative manner. A direct characterization of any ENT protein has been hindered due to difficulties in overexpression and obtaining pure recombinant protein. Methods: The equilibrative nucleoside transporter 7 from Arabidopsis thaliana (AtENT7) was expressed in Xenopus laevis oocytes to assess mechanism of substrate uptake. Recombinant protein fused to enhanced green fluorescent protein (eGFP) was expressed in Pichia pastoris to characterize its oligomeric state by gel filtration and substrate binding by microscale thermophoresis (MST). Results: AtENT7 expressed in X. laevis oocytes works as a classic equilibrative transporter. The expression of AtENT7-eGFP in the P. pastoris system yielded milligram amounts of pure protein that exists as stable homodimers. The concentration dependent binding of purine and pyrimidine nucleosides to the purified recombinant protein, assessed by MST, confirmed that AtENT7-eGFP is properly folded. For the first time the binding of nucleobases was observed for AtENT7. Significance: The availability of pure recombinant AtENT7 will permit detailed kinetic and structural studies of this unique member of the ENT family and, given the functional similarity to mammalian ENTs, will serve as a good model for understanding the structural basis of translocation mechanism for the family. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Nucleoside transporters are widely distributed in eukaryotes like protozoa, yeast, plants and mammals [1]. They mediate cellular uptake of nucleosides for nucleic acid synthesis in the salvage pathways in many cell types. In eukaryotes there are two major nucleoside transporter gene families: the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). ENT proteins are facilitated diffusion systems while the CNT proteins are cation symporters [2]. The ENT family is restricted to eukaryotes, whereas CNT family members are also found in eubacteria. In plants the members of the ENT protein family allow cellular nucleoside supply for the salvage pathway when nucleotide de novo synthesis activity is low [1].

⁎ Corresponding author at: Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Alberta T6G 2H7, Canada. E-mail addresses: [email protected] (C. Girke), [email protected] (E. Arutyunova), [email protected] (M. Syed), [email protected] (M. Traub), [email protected] (T. Möhlmann), [email protected] (M.J. Lemieux).

http://dx.doi.org/10.1016/j.bbagen.2015.06.003 0304-4165/© 2015 Elsevier B.V. All rights reserved.

In addition, ENT-mediated nucleoside supply can compensate nitrogen limitation [3]. No structural information is available for any member of ENT family. Topologically human, plant and yeast homologs of ENT family exhibit 11 predicted transmembrane helices with a cytosolic Nterminus, an extracellular C-terminus and a large hydrophilic loop in the region between transmembrane domains (TMD) 6 and 7, which has been experimentally confirmed for hENT1 and predicted for homologs [2,4–7]. In the model plant A. thaliana eight ENT homologs are present, five of which have been functionally characterized [7–9]. The substrate affinities have been also determined for Oryza sativa OsENT2 from rice [10]. Characterized plant ENT proteins transport nucleosides, deoxynucleosides and the phytohormon cytokinin riboside [1,10–12]. In contrast to mammalian ENTs, most plant homologs transport their substrates along a proton gradient, functionally resembling CNTs. Studies of A. thaliana ENT7 (AtENT7) expressed in yeast demonstrated the low nucleoside uptake dependence on the external pH [9], suggesting that AtENT7 could represent the classic equilibrative transporter and be functionally close to human ENTs. Furthermore, this protein shows high sequence similarity to drug uptake transporters, hENT1 and hENT2 [7] and, considering the

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fact that it is predicted to act as a true equilibrative transporter [9], AtENT7 may represent a relevant model to study equilibrative nucleoside transporters in vitro. Despite the importance of ENTs in a wide range of metabolic processes little is known about structural organization of this large protein family, in part due to the lack of expression system that would allow the preparation of large-scale quantities of stable protein [13]. The purification of complex membrane proteins in a functional state is often a challenging task and can be hampered by their tendency to aggregate irreversibly. Recently, the Saccharomyces cerevisiae ENT protein, FUN26, was successfully expressed and purified from S. cerevisiae. Liposome reconstituted FUN26 showed a proton-independent transport of nucleosides and nucleobases [4]. FUN26 is the only known ENT homolog in S. cerevisiae and is located at the vacuolar membrane where it is proposed to be involved in nucleic acid degradation and nucleoside delivery to the cytosol; the same functions have been shown for ENT3 from mice and ENT1 from A. thaliana [4,14]. While most ENTs in plants have been characterized to act in concentrative manner [7–10,15] here we demonstrated electroneutral nucleoside transport of AtENT7 expressed in X. laevis oocytes, confirming its close functional relationship to mammalian ENTs. Recombinant AtENT7 fused to enhanced green fluorescent protein (eGFP) was successfully overexpressed in P. pastoris. P. pastoris has proven to be a powerful expression host for complex membrane proteins due to several advantages compared to a bacterial system [16]. This eukaryotic expression system is capable of introducing post-translational protein modifications such as phosphorylation, glycosylation and lipidation, which can be important for the synthesis and stability of fully functional, recombinant membrane proteins [17]. Detergent-solubilized protein was purified using Immobilized Metal Affinity Chromatography (IMAC) and Fluorescence Size Exclusion Chromatography (FSEC) yielding milligram amounts of pure and stable transporter. Optimization of the purification protocol allowed us to characterize AtENT7 oligomeric state. Microscale thermophoresis (MST) binding studies clearly demonstrated the retained integrity of detergent solubilized AtENT7. The recombinant ENT7 protein will provide a strong foundation for structural studies of AtENT7 and a valid model for understanding the mechanistic basis of equilibrative nucleoside transport.

hotplate for 4.45 min at 450 °C. For the filling of electrodes and further details, see [20]. The calibration of pH-sensitive electrodes was carried out by changing the pH of extracellular solution by one pH unit (from 7.0 to 6.0). Oocytes, clamped at −40 mV, were perfused with different concentrations of adenosine (3, 10, 30 and 100 μM), respectively, added to HEPES (5 mM)-buffered OR2+ adjusted to either pH 7.0 or pH 6.0. For radioactive uptake measurements, AtENT3, AtENT7 or water injected oocytes were used (modified after [19]). For each experiment, seven injected oocytes were incubated in glass vials and washed twice with OR2+ (82.5 mm NaCl, 2.5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 1 mm Na2HPO4, 5 mm HEPES, pH 7.0). Subsequently 90 μl of 5 μM [3H]adenosine (37 GBq/mmol) in OR2+ solution were added. After 5 min, the radiolabeled medium was removed; oocytes were washed three times with ice-cold OR2+, separated and incubated in sodium dodecyl sulfate (10%). After incubation for 20 min radioactivity was counted in the presence of liquid scintillator in a Tri Carb 2500 TR (Packard, Canberra Industry, USA) scintillation counter. 2.3. Construction of AtENT7-eGFP The enhanced GFP, bearing the mutations F64L and S65T (eGFP) was amplified from pEGFP-N1 (Clontech) and cloned into pPICZA (Invitrogen), having C-terminal His8-tag and N-terminal tobacco etch virus protease (TEV) cleavage site and a four amino acid linker sequence. AtENT7 was amplified from the respective plasmid [9] with EcoRI and XhoI containing primers for N-terminally cloning to the GFP cassette in pPICZA-GFP. 2.4. Transformation and expression screening of AtENT7-eGFP in P. pastoris

A. thaliana ENT7 or ENT3 sequence was ligated into the oocyte vector pGEMHeJuel [18] as described previously [15]. Isolated plasmids were linearized with NotI, purified with the QIAquick PCR purification kit (Qiagen) followed by in vitro transcription (mMessage mMachineTM, Ambion http://www.ambion.de) and another PCR cleaning (RNeasy MinEluteTM-Cleanup, Qiagen). cRNA was stored at −70 °C.

pPICZA-AtENT7-eGFP plasmid was purified using maxi-prep (Qiagen), after which 20 μg of DNA was linearized overnight at 37 °C with SacI (Fermentas, USA), incubated with 80 μl of electrocompetent P. pastoris GS115 cells on ice and electroporated using a model 2510 electroporator (Eppendorf-Netheler-Hinz, Hamburg, Germany) at 2.5 kV, 25 μF, 100 Ω. Cells were plated on YPDS (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol) media containing 100 μg/ml zeocin, and incubated at 30 °C until colonies appeared (24–48 h). From this plate, 48 colonies were picked and spotted on fresh BMMY plates (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% YNB (yeast nitrogen base), 4 × 10−5 M biotin, 0.5% methanol) using a grid. As a negative control, two colonies of untransformed GS115 cells were used. Two clones of mouse PEMT, that were previously identified as high expressing clones by our group [16], were included as positive controls for expression. Plates were incubated at 30 °C and imaged every 24 h for 4 days using an ImageQuant LAS4000 imager, equipped with blue light (GE Healthcare, USA). All exposures were taken at 1/8th of a second. The mean gray values (MGV) of each colony were determined for quantification of the intensity of expression using ImageJ software [21].

2.2. Electrophysiology and radioactive uptake studies

2.5. Expression and purification of AtENT7-eGFP

The electrophysiological experiments were performed as described in a previous report [18]. Oocytes were injected with 23 ng AtENT3cRNA or AtENT7-cRNA using glass micropipettes and a microinjection device (Nanoliter 2000; World Precision Instruments http://www. wpi-europe.com). Control oocytes were injected with an equivalent volume of diethylpyrocarbonate (DEPC)-H2O. The electrophysiological experiments were performed using an AxoClamp 2B amplifier (Axon Instruments http://www.axon.com) equipped with current- and doublebarreled ion selective microelectrodes [19] connected to the headstages of the amplifier. For pH-sensitive electrodes, two borosilicate glass capillaries (1 and 1.5 mm in diameter) were twisted together and pulled to a micropipette. The tips of the micropipettes were backfilled with a mix of tri-N-butylchlorsilane and pure carbon tetrachloride and baked on a

The high expressing clone of AtENT7-eGFP identified on the screen plate was inoculated in 100 ml BMGY media (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% YNB, 4 × 10− 5 M biotin, 1% glycerol), containing 100 μg/ml of Ampicillin and grown at 28 °C, 300 rpm until OD600 of 7 as a starting culture. Then 6 l of BMGY with 100 μg/ml of Ampicillin were sub-inoculated with the starting culture and grown for 14–18 h at 28 °C, 250 rpm to OD600 of 5–10 (the log phase of the growth). The protein expression was induced by transferring the cells in BMMY media by pelleting and resuspension in an equal volume of BMMY. Fluorescence measurements were taken every 12 h to monitor expression. Cells were harvested from 5 ml of media (1500 g, 10 min) and resuspended in 200 μl of PBS in a 96well plate (Costar, USA). Fluorescence was measured using a

2. Materials and methods 2.1. Construction of oocyte expression vector

C. Girke et al. / Biochimica et Biophysica Acta 1850 (2015) 1921–1929

FluoroSTAR fluorescence plate reader with the excitation wavelength of 488 nm and emission wavelength of 509 nm with a gain of 800. When the fluorescence reached the maximum level (48 h of expression) the cells were harvested (1500 g, 15 min), resuspended in 50 mM Tris– HCl, pH 8, 150 mM NaCl, 5% glycerol buffer containing DNase and Protein Inhibitor Cocktail Tablets (Roche, USA) in the ratio of 1:4 (weight:volume) and lysed using a TS model cells disruptor (Constant Systems Ltd., United Kingdom) at 40 kpsi. Cell debris was removed by two consecutive centrifugation steps: the first at 3000 g for 10 min, followed by a second centrifugation step at 16,000 g for 20 min. Lastly, membranes were isolated by ultracentrifugation at 100,000 g for 2 h. The membranes were homogenized in 50 mM MES, pH 6.0 containing 150 mM NaCl, 10 mM Imidazole, 20% glycerol and solubilized with 1% n-Dodecyl β-D-Maltopyranoside (DDM) (Anatrace, USA) for 1 h at 4 °C. Insoluble material was pelleted at 100,000 g for 30 min and the protein sample was dialysed against 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 10% glycerol buffer for 2 h at 4 °C to adjust the pH to 8.0 for the optimal binding properties for metal ion chromatography. The supernatant was applied on Ni-NTA column (Qiagen, Ontario, Canada) and the protein was eluted with step gradient: 100–500 mM imidazole in 50 mM MES, pH 6.0, 150 mM NaCl, 20% glycerol, 0.1% DDM buffer. The purity of fractions corresponding to AtENT7-eGFP was assessed with SDS-PAGE (in-gel fluorescence and Coomassie staining) and FSEC on Superdex 200 (10/30) column, equilibrated with 50 mM MES, pH 6.0, 300 mM NaCl, 10% glycerol, 0.1% DDM. After the gel-filtration the protein samples were flash-frozen and kept at −80 °C.

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free eGFP was subjected to SEC on Superdex 200 (10/30) column, equilibrated with the binding buffer. 3. Results 3.1. AtENT7 exhibits the properties of classic equilibrative transporters

The pPICZA-GFP plasmid was transformed into electrocompetent P. pastoris GS115 cells and the protein was expressed for 48 h following the above mentioned protocol. The cells were resuspended in 50 mM Tris–HCl, 150 mM NaCl, 5% glycerol buffer, containing DNase and Protein Inhibitor Cocktail Tablets (Roche, USA) and lysed as described above. Cell debris was removed at 3000 g for 10 min and then at 16,000 g for 20 min. The supernatant was loaded on Ni-NTA column and the protein was eluted in the same buffer with increasing concentration of imidazole (100–500 mM). The purified protein was flashfrozen and kept at -80 °C.

Despite their name, most plant ENTs catalyze proton–nucleoside cotransport, thus functionally resembling concentrative nucleoside transporters (CNTs) [8,9,15]. However, in the course of the uptake studies of AtENTs expressed in yeast [9] we observed that AtENT7, in contrast to e.g. AtENT3, showed no alterations in nucleoside uptake when the pH of the incubation medium was changed. Therefore, we speculated that AtENT7 might function as a diffusion carrier independent from transmembrane proton gradients. To confirm this hypothesis AtENT7 and AtENT3 were expressed in X. laevis oocytes for electrophysiological and uptake studies. AtENT3 was used as a control because the clear pH dependence of nucleoside transport after heterologous expression of this gene in yeast cells was published previously [9]. To determine whether both expressed transport proteins were functional after injection of the corresponding cRNA, uptake of radiolabeled adenosine was measured. The measured uptake rates were almost identical (40 pmol h−1 oocyte−1) for both transporters indicating their functionality in oocytes (Fig. 1A). For the electrophysiological studies, the two-electrode voltage clamp technique was applied. The transport of adenosine by recombinant AtENT3 expressed in X. laevis oocytes was clearly electrogenic at Vh = −30 mV using both pH 6.0 and 7.0 for bath solutions. The intracellular proton concentration rose in a substrate dependent manner (Fig. 1B). In contrast, AtENT7 recombinantly expressed in the same batch of oocytes evoked currents only at background noise level, indicative of an equilibrative mode of transport (Fig. 1C). In addition, no alteration of the intracellular proton concentration could be measured in response to supplied adenosine for AtENT7 (Fig. 1C). Thus, AtENT7 is the first plant family member that transports nucleosides in an equilibrative mode and thus represents a classical ENT. Having 22% of sequence identity and 43% of similarity to both, hENT1 and hENT2, this plant protein represents a suitable model for structural and biochemical characterization of ENT family and therefore was chosen for overexpression in the P. pastoris system.

2.7. Assessment of nucleoside binding to AtENT7-eGFP using microscale thermophoresis

3.2. Expression of AtENT7 in the P. pastoris system

2.6. Purification of free eGFP from P. pastoris

Prior the binding experiments AtENT7-eGFP, purified on the Ni-NTA column, was subjected to size exclusion chromatography (SEC) on Superdex 200 (10/30) equilibrated with 50 mM sodium phosphate, pH 6.0, 100 mM NaCl, 10% glycerol, 0.1% DDM (binding buffer). The fluorescence was measured for each fraction collected and the samples with the highest fluorescence and protein concentration were pooled and used for MST studies. The measurements were performed on a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies, Germany). AtENT7-eGFP was used in a concentration of 65 nM for all binding experiments. Stock solutions of ATP, nucleosides or nucleobases were prepared followed by serial dilutions in 50 mM sodium phosphate, pH 6.0, 100 mM NaCl, 10% glycerol, 0.1% DDM buffer. Monolith NTTM Standard Treated Capillaries (NanoTemper Technologies, Germany) were used. In order to find the best thermophoretic setting, we analyzed the binding of nucleosides to AtENT7-eGFP at different infrared laser powers. The best signal to noise ratio was obtained by using 20% of the maximum heating power, whereas stronger excitation resulted in higher aggregation level. Thus, all measurements were performed at 70% LED and 20% MST power; laser-on time was 30 s and 5 s final laser-off time for back diffusion. As negative controls, the binding of AtENT7-eGFP to the same concentration range of ATP, as well as the binding of nucleosides and nucleobases in the same concentration range to 65 nM of free eGFP were assessed. Prior to the binding studies

The ENT7 gene from A. thaliana was cloned into pPICZA plasmid, containing C-terminal eGFP and His8-tag, separated by tobacco etch virus protease (TEV) cleavage site with a small peptide linker (GGGS). Initial selection for genomic integration of AtENT7-eGFP was performed on Zeocin-containing YPDS plates. In order to identify the highest expressors among the transformants, a rapid plate-screening method, developed by our group was employed (Fig. 2) [16]. Clones from YPDS plate were translocated onto BMMY plate to induce protein expression (Fig. 2A). Two untransformed clones of GS115 cells were used as negative controls, two clones expressing membrane protein phosphatidylethanolamine N-methyltransferase (PEMT), previously demonstrated by our group to have a high level of protein expression [16] were used as a positive control (Fig. 2A). The fluorescence of each colony was quantified (Fig. 2B). The AtENT7-eGFP clone at position 14 with the highest fluorescent signal was chosen for optimization of growth conditions and further large-scale expression. 3.3. AtENT7 purification After harvesting and lysing the cells, differential centrifugation was performed. As shown in Fig. 2C, these steps removed the majority of aggregates, degradation products and most of free eGFP that commonly co-purify with eGFP-fused proteins.

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Fig. 1. Uptake and electrophysiology assays of AtENT7 and AtENT3 expressed in X. laevis oocytes. A. Radioactive uptake of adenosine by X. laevis oocytes expressing AtENT7 or AtENT3. Data represent the mean from seven oocytes ± SE. B. Intracellular pH recordings and membrane currents with voltage steps between −100 and +20 mV for obtaining current voltage relationships measured in an oocyte injected with 23 ng AtENT3-cRNA during brief applications of adenosine (3–100 μM) at pH 7.0 and 6.0. Reproduced from [15]. C. Intracellular pH recordings and membrane currents after injection of an oocyte with 23 ng AtENT7-cRNA. All oocytes were taken from the same batch.

solubilized. 1% was the best working concentration; increasing the amount of DDM did not result in improved solubilization of AtENT7eGFP. The optimal time for solubilization was 1 h with longer and shorter incubation times resulting in less solubilized protein. The major advantage of fused eGFP was the possibility to assess the aggregation behavior of solubilized transporter prior to purification with the help of fluorescent size-exclusion chromatography (FSEC). FSEC is an effective screening technique that also allows examination of stability and the approximate molecular weight of the GFP-fused protein [23,24]. Different buffers were evaluated and the impact of pH on the oligomeric state of AtENT7-eGFP, in particular, was assessed. FSEC profiles of the crude membranes solubilized in the buffers with three different pH values (pH 6, 7 and 8) and the same concentration of DDM (1%) are presented in Fig. 3A. Adjusting the pH of the buffer resulted in a clear transition of solubilized protein from an aggregated state to a homogeneous state. At pH 8.0 only large oligomers were observed on FSEC profile (VE = 8.8 ml, N 1000 kDa), while at pH 7 a large proportion of oligomers (VE = 11 ml, 398 kDa) and some dimers were detected. Yet, at pH 6.0 FSEC resulted in predominantly dimeric state (VE = 12.4 ml, 210 kDa; MW of AtENT7-eGFP is 71 kDa, MW of DDM micelle in SEC buffer (Dynamic light scattering calculated) is 40 kDa) with almost no aggregation. Thus, pH 6.0 was demonstrated to be the optimal condition for solubilization and further purification. Importantly, in the course of purification prior to the Ni-NTA chromatography, the pH of the solubilized protein was brought up to 7.5 to provide the optimal conditions for binding to the Ni resin. The elution of AtENT7-eGFP with increasing concentration of imidazole was conducted again at pH 6.0. It is worth mentioning that the pH value during the solubilization step was crucial to prevent aggregation. If the protein was solubilized at pH 8.0, the lowering of the pH to 6.0 at the elution step or during the FSEC was not able to shift the protein from aggregated to dimeric state. This purification strategy led to an increased amount of purified AtENT7-eGFP, with little contamination (Fig. 3B) and the final yield being 2 mg/l of yeast culture. The SEC profile of the purified transporter reveals that AtENT7-eGFP exists as a dimer in detergent solution. To confirm that eGFP did not promote dimerization of AtENT7-eGFP the SEC of purified eGFP domain alone was conducted. eGFP eluted as a monomer (VE = 16.2 ml, 25 kDa) (Fig. 3C) corroborating the fact of the dimeric nature of AtENT7. It is known that the presence of relatively large fusion partner can stabilize the protein [25]. To examine if AtENT7 can remain stable in a detergent solution, eGFP was cleaved off with TEV protease. The conditions for 100% cleavage of fusion protein were optimized and set up at 2 h, 4 °C, 1:10 TEV to protein ratio. The TEV protease, eGFP and the traces of uncut protein were removed with the help of Ni-NTA chromatography. The stability and oligomeric state of obtained protein was assessed by SEC (Fig. 3D). The purified AtENT7 eluted in a peak corresponding to the molecular weight of the dimeric state (VE = 12.8 ml, 165 kDa, MW of AtENT7 is 45.7 kDa), providing further evidence to the fact that AtENT7 was a dimer in detergent solution. It is worth mentioning, that after storage at − 80 °C for several months and a freeze–thaw cycle, the final protein sample yielded a single symmetrical peak with no aggregates on the SEC profile (data not shown) and thus revealed stability. 3.4. Nucleoside and nucleobase binding ability of purified AtENT7-eGFP

The choice of detergent is vital for isolation and purification of membrane proteins [22]. The extent of solubilization and the stability of the solubilized protein depend on the nature of detergent used and its concentration; therefore a broad screen using different detergents for solubilization was performed. Small aliquots of isolated membrane fraction were solubilized for 1 h in a buffer, containing detergents in a concentration of 100 × CMC (Critical Micelle Concentration). Insoluble particles were removed by centrifugation at 100,000 g for 30 min and the amount of solubilized protein in the supernatant was evaluated by fluorescence measurements (Table 1). DDM had the best solubilizing properties among all tested detergents with 51% of AtENT7-eGFP

To assess the substrate binding properties of the purified transporter, we used microscale thermophoresis (MST), a novel and powerful technique that enables the direct monitoring of the protein substrate complex formation, given that one partner carries a fluorescent label [26]. In our experiments the concentration of AtENT7-eGFP was kept constant at 65 nM, while the concentration of substrates was varied. After a short incubation, the samples were loaded into standard treated glass capillaries and MST analysis was performed. The complex formation led to a fluorescent signal that enabled the measurement of affinities (Kd). True binding events can be separated from aggregation and other artifacts

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Fig. 2. Induction plate expression screening of AtENT7-eGFP. A. Clones expressing AtENT7-eGFP were spotted onto BMMY plates and incubated for 72 h at 30 °C. P. pastoris GS115 was spotted onto positions 1 and 2 as a negative control. Clones of mouse PEMT described as high expressors were included as positive controls in positions 3 and 4. B. Mean gray values of the yeast colonies from A. C. SDS-PAGE in-gel fluorescence of centrifugation step samples. Lane 1 — molecular weight markers, kDa; lane 2 — the pellet after centrifugation for 10 min at 3000 g; lane 3 — the pellet after centrifugation for 20 min at 16,000 g; lane 4 — the membrane pellet after centrifugation for 2 h at 100,000 g; lane 5 — the supernatant after centrifugation for 2 h at 100,000 g.

by analyzing the fluorescent signal during the “laser on time” cycle. The fluorescence signal during that time was smooth, strongly indicating the absence of aggregates. To ensure that the binding is transporter-specific, purified eGFP was used as a negative control. Binding of all four tested nucleosides (adenosine, guanosine, uridine and cytidine) to AtENT7eGFP resulted in a clear and strong response in fluorescence signal that was dependent on nucleoside concentration (Fig. 4 A–D). Control measurements with eGFP revealed neither nucleoside nor nucleobase dependent fluorescence signals (Figs. 4 and 5). The binding of ATP to AtENT7eGFP also did not result in fluorescence signal (Fig. 4 E). The calculated Kd for the interaction between AtENT7-eGFP and adenosine was 1.12 ± 0.19 μM, for guanosine — 8.11 ± 1.74 μM, for cytidine — 87.60 ± 15.50 μM and for uridine — 16.60 ± 2.20 μM. These results are in good agreement with previously calculated nucleoside transport affinities for AtENT7 expressed in yeast cells (Table 2). In addition, for the first time for AtENT7 we were able to demonstrate the binding to nucleobases – adenine, guanine, cytosine and uracil – with apparent Kd values of

Table 1 The solubilization properties of different detergents towards AtENT7-eGFP. Solubilization was conducted for 1 h at 4 °C, the insoluble membranes were removed by centrifugation at 100,000 g for 30 min. The fluorescence measurements of the supernatant were performed before and after solubilization. Detergent

Fluorescence before solubilization [RFU]

Fluorescence of supernatant after solubilization [RFU]

Solubilization efficiency [%]

DDM TX-100 CYMAL-1 OG DM LDAO H2O

57,865 40,683 43,435 50,226 59,590 55,364 58,471

29,416 15,148 12,457 10,677 2158 1560 968

51 37 29 21 3.6 2.8 1.7

18.8 ± 0.92 μM, 13.50 ± 1.87 μM, 18.9 ± 4.55 μM and 13.60 ± 3.6 μM respectively (Fig. 5A–D).

4. Discussion Equilibrative nucleoside transporters are membrane proteins ubiquitously found in eukaryotes. They play the key role in nucleoside uptake and provide substrates for salvage pathways of nucleotide synthesis. Human pathogenic protists typically lack enzymes involved in the de novo biosynthesis of purines [27] and hence purine nucleosides need to be imported to cope with the high demand on RNA and DNA synthesis. Moreover, in human ENTs inhibitor recognition and the transport of an array of therapeutically important inhibitors, nucleoside derivates or other permeants are at the focus of current research [28]. Despite the profound ENT significance for nucleoside physiology and pathophysiology, our understanding of structural determinants and functional features of this transporter family is still limited. This is explained by the fact that the structural and functional characterization of ENTs requires milligram amounts of protein and is hampered by the inability to overexpress and purify stable and functional transporters [13,28,29]. Recently FUN26, the sole nucleoside transporter that is present in S. cerevisiae vacuoles, was successfully expressed and purified from S. cerevisiae [4], however, the heterologous expression and purification of any ENT has never been achieved. In this study using the P. pastoris system, we were able to demonstrate high yield expression of AtENT7 fused to eGFP (2 mg purified protein per liter of yeast cells) and, most importantly, purify homogeneous and stable protein, which was able to bind to its substrates. Homogeneity and stability of protein samples are prerequisites required for successful structural studies and functional assays. The fusion of the protein of interest to eGFP had multiple applications. This construct allowed us to rapidly screen many colonies while choosing the best

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Fig. 3. Purification of AtENT7. A. FSEC profiles of yeast crude membranes with expressed AtENT7-eGFP solubilized in buffers with different pH values. B. Absorbance and fluorescence profiles of SEC of AtENT7-eGFP purified at pH 6. Inset: SDS-PAGE of AtENT7-eGFP Ni-NTA purification. Lane 1 — molecular weight markers, kDa; lane 2 — elution with 100 mM of imidazole; lane 3 — elution with 500 mM of imidazole. C. Absorbance and fluorescence profiles of SEC of eGFP. D. SEC of AtENT7 after removal of eGFP and TEV. Arrows indicate the standards for Superdex 200 (10/30). 1 — Thyroglobulin, 9.63 ml (MW, 670 kDa; Stokes radius 86 Å), 2 — IgG, 12.95 ml (MW 158 kDa; Stokes radius 51 Å), 3 — Ovalbumin, 15.5 ml (MW 44 kDa; Stokes radius 28 Å), 4 — Myoglobin, 17.3 ml (MW 17 kDa; Stokes radius 19 Å) Inset: SDS-PAGE of AtENT7-eGFP TEV cleavage. Lane 1 — molecular weight markers, kDa; lane 2 — uncleaved protein; lane 3 — AtENT7 (the flow-through from Ni-NTA purification of cleaved sample); lane 4 — eGFP and TEV protease (the elution from Ni-NTA column).

expressors and to control the expression level, by monitoring the fluorescence during the cell growth. eGFP also served as a “folding indicator”. The latter was possible due to the fusion of eGFP to the Cterminus of the transporter. Recently published preliminary studies of AtENT7 homologs AtENT1 and AtENT3 as model targets to test the expression of membrane proteins in a cell free system with fused eGFP, revealed a good potential of such approach and showed the correct folding of the expressed fusion protein [30]. The high fluorescence level of expressed AtENT7-eGFP as well as the FSEC profile of purified protein suggested that the purified transporter was folded correctly. FSEC is a useful tool to monitor a monodispersity; a folded protein typically yields a single symmetrical peak (see Fig. 3B), while polydisperse, unstable or unfolded protein yields multiple, asymmetrical peaks [31]. Furthermore, the facts that after a freeze thaw cycle of AtENT7-eGFP a single symmetrical peak was observed on the SEC profile suggested high protein stability. The binding properties of transporter were also not affected by freezing.

Optimization of purification conditions enabled us to obtain the pure AtENT7-eGFP in dimeric state. SEC of free eGFP eluted in a monomeric form eliminated the possibility that eGFP triggered dimerization of fusion protein and allowed us to conclude that AtENT7-eGFP dimer formation appears to be solely the feature of transporter. Several other research groups also reported that eGFP exists as monomer in solution [32,33]. The fusion of a protein of interest to a partner often leads to improved stability. However, the most crucial factor for further structural and functional characterization is the recovery of the target protein upon tag cleavage. The eGFP was successfully cleaved off from AtENT7 with TEV protease and SEC resulted in a stable transporter with no aggregation. The calculated molecular weight of the eluted protein also suggested that AtENT7 existed as a dimer in detergent solution. The successful overexpression and purification of ENT family members will pave the way to more detailed functional characterization of substrate specificities of the transporter. This task was proved to be

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Fig. 4. The binding affinities of AtENT7-eGFP to nucleosides and ATP as determined by MST. The difference in normalized fluorescence Fnorm [‰] is plotted for analysis of MST TJump. (A — adenosine, B — guanosine, C — cytidine, D — uridine, E — ATP). eGFP was used as negative control and shows no binding to any nucleosides. The plots are representative of three independent experiments and Kd is shown as mean and standard deviation.

challenging with ENTs expressed either in yeast or in oocytes due to the presence of other membrane proteins/transporters and often their overlapping functional roles. To confirm the correct folding and unimpaired binding abilities of AtENT7 to its substrates, the MST technique was employed. In addition to the determination of binding properties, this method allows for assessing the level of potential protein aggregation. The smooth curves of the fluorescence signal over time when the IRLaser was on and no change in back diffusion after turning the IR-Laser off were indicative of no aggregation of the protein sample [26,34]. Although MST does not permit investigating true transport, it provides a robust and fast way to determine the protein integrity, measure

substrate interactions and assess affinities. It was successfully used for substrate binding affinity determination for plant nitrate transporter NRT1.1 [35] and the obtained value of Kd (1 mM) was comparable to the affinity of NRT1.1 synthesized in X. laevis oocytes (KM ≈ 4 mM) [36], giving the rationale of using this technique for assessment of functionality. The binding affinities of AtENT7-eGFP for nucleosides were found to be in the concentration range of previously identified substrate affinities for the protein expressed in yeast cells (Fig. 4; Table 2) [9] with slightly lower Kd value for adenosine (1.12 μM) compared to the previously reported affinity (KM = 9.8 μM). The binding of nucleobases (adenine, uracil, cytosine and guanine) was demonstrated for the first time

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Fig. 5. The binding affinities of AtENT7-eGFP to nucleobases as determined by MST. The difference in normalized fluorescence Fnorm [‰] is plotted for analysis of MST T-Jump. (A — adenine, B — guanine, C — cytosine, D — uracil). eGFP was used as negative control and shows no binding to any nucleobase. The plots are representative of three independent experiments and Kd is shown as mean and standard deviation.

for AtENT7 with the affinities ranging from 10 to 20 μM (Fig. 5). However, our earlier AtENT7 studies suggest that nucleobases do not significantly inhibit nucleoside transport [9], which may imply independent binding sites. High affinities towards nucleosides and nucleobases were also observed for FUN26 reconstituted into proteoliposomes (0.19–0.32 μM) [4], whereas hENTs transport nucleosides and especially nucleobases with lower affinities (μM–mM range) [37, 38], although it is worth noting that binding and transport assays have never been conducted with any purified hENT protein. In contrast to mammalian ENTs, most plant homologs transport their substrates along a proton gradient. Studies of AtENT7 expressed in yeast demonstrated that nucleoside uptake was independent of the external pH [9], suggesting that AtENT7 could represent the classic equilibrative transporter and be functionally close to mammalian ENTs. In this report, we employed an electrophysiological assay to confirm this hypothesis. The fact that we were able to demonstrate adenosineTable 2 Kd values of AtENT7-eGFP purified from P. pastoris for nucleosides (MST data are presented as means ± SD. n ≥ 2) and KM values of AtENT7 overexpressed in S. cerevisiae for nucleosides [9].

Adenosine [μM] Guanosine [μM] Cytidine [μM] Uridine [μM]

Kd (AtENT7-eGFP expressed and purified from P. pastoris)

KM (AtENT7 expressed in S. cerevisiae)

1.12 ± 0.19 8.11 ± 1.74 87.60 ± 15.50 16.60 ± 2.20

9.8 9.4 40 13.4

induced proton import in oocytes expressing AtENT3 justified the applicability of this assay for electrogenic transport studies. For AtENT7 expressed in oocytes we showed non-electrogenic adenosine transport, confirming our previous observations. Thus, AtENT7 was demonstrated to act as a equilibrative nucleoside transporter in comparison to other ENT proteins from plants and protists [10,15,39,40]. This functional resemblance to mammalian ENTs makes this plant protein a promising model for deciphering structural information and functional details of uptake and the high yield purification is a crucial step towards these characterization efforts. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgments We thank Dr. Cory Brooks and Melissa Morrison for helping with AtENT7 cloning. The work of Christopher Girke and Dr. Torsten Möhlmann was supported by DFG-grant MO 1032/3-2 and IRTG1830. Research in MJL's group is supported by grant MOP-93557 from the Canadian Institutes of Health Research, as well as a grant from the Canadian Foundation for Innovation. MS was supported by a summer studentship from the Natural Sciences and Engineering Research Council-supported International Research Training Group in Membrane Biology. MJL is an Alberta Innovates Health Solutions Scientist.

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