The FPS1 gene was tagged at its 3' end with a sequence encoding the HA3 epitope to permit immunodetection; the carboxy-terminal threonine residue was replaced with the amino acid sequence: SGRIF YPYDVPDYA G YPYDVPDYA G YPYDVPDYA AQCGR. The HA3-tag sequence is underlined. The construct was expressed from the TPI promoter in the 2 μ pYX212 vector (Novagen; now discontinued) which contains the URA3 selection marker. The gene was cloned into the Bam H1 and Hin dIII sites and the vector transformed into S. cerevisiae TM6*, KOY.PK2-1C82 and BY4741. For the S. cerevisiae 954 VW K70 strain the construct was expressed from the TPI promoter in the 2 μ pYX222 vector (Novagen; now discontinued), which contains the HIS3 selection marker. Genes encoding human A2aR and CNR2 receptors were tagged as above at the 3' end with the HA3-tag and at the 5' end with a sequence encoding the S. cerevisiae α factor secretion signal: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAGS. The constructs were expressed from the TPI promoter in both the pYX212 and pYX222 vectors. The pCU426 GFP construct was the generous gift of Dr Arle Kruckeberg.
Yeast strains and growth conditions
The constructs cloned into plasmids pYX212 and pCU426 were transformed into S. cerevisiae strains KOY-TM6*, referred to as TM6* , KOY.PK2-1C82 (MATa, ura 3-52, MAL 2-8
SUC2)  and BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0). Constructs cloned into plasmid pYX222 were transformed into S. cerevisiae strain 954 VW K70 (MATα his3 SUC2 GAL MAL 2-8
). Yeast transformants were initially grown on YNB agar plates lacking uracil or histidine to select for plasmid retention. For the TM6* Fps1 cultivations, 5 mL cultures were inoculated and grown for 72 h prior to being used to inoculate 50 mL cultures, which were grown for a period of 24 h and used to inoculate cultures in 2.5 L to a final OD610 of 0.05. For the wild-type cultivations, 100 mL cultures were grown for 24 h before inoculating the bioreactors to a final OD610 of 0.05. Yeast cells were cultured in 2.5 L 2 × CBS medium , with 2 % glucose as the sole carbon and energy source. For growth of TM6* with ethanol in the medium, 7 g L-1 ethanol were added to the bioreactor together with glucose just before the inoculation. The pH of the cultures was maintained at pH 5 using 1 M NaOH. Polypropylene glycol P2000 was added as antifoam (100 μL L-1). Agitation and aeration of the cultures were set at 1000 rpm and 0.5 vol vol-1 min-1, respectively whilst the temperature was set at either 20°C or 30°C. The control of the pH, agitation, aeration and temperature was maintained online. Gas evolution was monitored on-line (type CP460 O2/CO2, Belach Bioteknik AB).
For glucose fed-batch cultivations, the wild-type S. cerevisiae strain 954 VW K70 was cultured at 30°C, pH 5. 100 mL cultures were grown for a period of 24 h and used to inoculate cultures in 2.5 L bioreactors to a final OD610 of 0.1. Yeast cells were cultured in 2.0 L YNB medium (1.7 g L-1 YNB (Q-Biogene), 5 g L-1 (NH4)2SO4) with 1 % glucose until glucose was depleted, and then grown with a feed of 500 mL 10 % glucose at a flow rate of 20 mL h-1. Polypropylene glycol P2000 was added as antifoam (100 μL L-1). Agitation of the cultures was set at 600 rpm and airflow of 0.6 L min-1. All cultivations were at least duplicated. We have previously developed a series of simple functional in vivo assays for aquaporins in yeast  (and references therein), and hence were able to confirm that the Fps1 was active under these conditions. Fps1 activity in wild-type yeast was previously reported , and there were no indications of any differences in this study.
BY4741 or TM6* transformed with the GPCR constructs were initially grown on YNB plates lacking uracil to verify plasmid retention. Single colonies were then used to inoculate 50 mL 2 × CBS medium and cultured for a period of 24-48 h. These pre-cultures were then used to inoculate 500 mL 2 × CBS medium in a bioreactor to a final OD610 nm of 0.05 and cultured until glucose depletion was detected using a Accu-Chek active glucose analyser (Roche diagnostics, UK) according to the manufacturer's instructions. Cells were harvested by centrifugation at 5, 000 × g, 4°C, 5 min.
BY4741 or TM6* transformed with the GFP construct were grown on YNB plates lacking uracil to verify plasmid retention. Single colonies were then used to inoculate 50 mL YNB medium and cultured for a period of 48 h. These seed cultures were then used to inoculate fresh 50 mL YNB medium in shake-flasks containing 0 or 1 mM CuSO4 to a final OD610 of 0.15 and cultured for a period of 24 h. Cells were harvested by centrifugation at 5, 000 × g, 4°C, 5 min.
Protein, cell dry weight and extracellular substrate determination
Samples for optical density measurements were taken periodically to measure growth. For the wild-types and ethanol-phase TM6* cultures, additional samples for subsequent protein, dry weight, and extracellular substrate/product analysis were taken out at early, mid and late glucose phase (1-3-Glc) corresponding to a glucose concentration of 1.5 %, 1.0 % and 0.8 % respectively, 4-Glc (when the glucose is depleted), and 5-EtOH and 6-Stat during the ethanol and stationary phases, 6.5 h, and 18.5 h after glucose depletion, respectively. The same procedure was performed for the TM6* cultures but also at additional time points. Therefore Glc 1-3 correspond to glucose concentrations above 1.5 %, 1.5-0.5 % and below 0.5 %, respectively. 4-Glc corresponds to glucose exhaustion and the stationary phase samples, 5-Stat, for the TM6* cultures are samples taken at least 10 h after glucose exhaustion. For extracellular substrate analysis, samples (2 × 1 mL) were collected, and the supernatant recovered by centrifugation (13, 000 × g for 1 min) and stored at -20°C prior to analysis with Boehringer Mannheim GmbH kits (Food Diagnostics, Stenungsund, Sweden) according to the manufacturer's instructions. Dry weight calculations were performed by harvesting 2 × 5 mL samples in pre-weighed, desiccated sample vials, and the cell pellet recovered by centrifugation at 5,000 × g for 5 min, at 4°C. The cells were subsequently washed twice with 5 mL ice-cold MilliQ water, and recovered by centrifugation, as above. The pellets were then dried for 24 h at 110°C, and stored in a desiccator prior to being weighed. Samples for protein analysis (1-4 × 50 mL) were harvested and the cell pellet recovered by centrifugation at 5,000 × g for 5 min at 4°C and stored at -20°C.
Membrane preparation and immunoblots
Analysis of Fps1 protein yields were performed on both the total cellular extract and the total membrane fraction. Cells were suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 0.5 mM EDTA, 5 % glycerol and mixed with glass beads at a 1:1:1 ratio. The cells were agitated in a FastPrep (Q-Biogene) at a speed of 6.5, 3 × 20 s with a 60 s incubation on ice between the pulses. Unbroken cells were recovered by centrifugation at 500 × g for 10 min at 4°C, and the supernatant further clarified at 10,000 × g for 30 min at 4°C. The total membrane fraction was collected from this clarified supernatant by ultra-centrifugation at 100,000 × g for 90 min at 4°C. The protein concentration was estimated using a BioRad Protein Assay Kit with bovine serum albumin as a standard. Immunoblotting was carried out with 35 μg of crude cell extract and 75 μg of total membrane fraction loaded on a 7.5 % SDS polyacrylamide gel and separated at 65 V through the stacking gel and 140 V through the separating gel for approximately 1 h. Proteins were transferred to a nitrocellulose membrane and blocked with PBS containing 5 % milk for 1 h before being probed with a mouse monoclonal HA antibody (1:1,000) overnight in PBS containing 5 % milk. After washing with PBS containing 5 % milk the membrane was incubated with a secondary goat anti-mouse IgG HRP conjugated antibody (1:2,500) for 1 h in PBS containing 5 % milk. The membrane was then washed in PBS containing 5 % milk; followed by PBS containing 0.1 % Tween-20 and finally with PBS. The blots were developed with an ECL Plus Western Blotting Detection Kit (Amersham Pharmacia) according to the manufacturer's instructions, visualised using the Image Reader LAS-100 (Fujifilm), and quantified using Multi Gauge 3.0 (Fujifilm). Either total extract or the membrane-bound fraction isolated from Sample 2 of a 30°C, pH 5 wild-type culture  in YNB medium was used as an internal standard, which is the yield of Fps1 during the glucose growth phase of cells cultured at 30°C, pH 5 when the OD610 was 1.5-3.0 and residual glucose levels were 2-5 g/L. All signals were below saturation and related to the signal of the internal standard.
Radioligand binding on wild-type and TM6* membranes containing recombinant human A2aR and CNR2
Cells were washed once with ice-cold breaking buffer (50 mM sodium phosphate buffer pH7.4, 100 mM NaCl, 5 % glycerol, 2 mM EDTA, 1 mM PMSF) and suspended to 30 % wet weight. 0.5 mm glass beads were added to the cell suspension at a 1:1 ratio and cells broken at 4°C using a FastPrep24 cell disrupter (MP Biomedical) with 8 cycles of 30 s shaking and 30 s incubation on ice. Unbroken cells/cell debris were separated from the membrane suspension by centrifugation (3000 × g, 5 min, 4°C). Total membranes were then recovered at 100,000 × g, 45 min, 4°C and suspended in membrane buffer (50 mM Tris pH 8.0, 120 mM NaCl, 20 % glycerol, 1 mM PMSF) using a dounce homogenizer. Membrane proteins were quantified following the BCA method (Pierce, Rockford, IL, USA), using BSA as a standard and snap frozen in liquid nitrogen prior to storage at - 80°C.
Saturation ligand binding assays were performed for membranes containing human A2aR with [3H]ZM241385 and ZM241385 (10 μM) ligands in 50 mM Tris HCl pH 7.4, 1 mM EDTA, 10 mM MgCl2, and incubated at 25°C for 1 h. In the case of human CNR2, [3H]CP55940 and CP55940 (50 μM) in 50 mM Tris HCl pH 7.4, 2.5 mM EDTA, 5 mM MgCl2, 0.5 mg ml-1 BSA were incubated at 30°C for 1.5 h. For each experiment, 10 μg of membrane protein were incubated in triplicate with increasing concentrations of radioligand until equilibrium was reached (total binding conditions). For non-specific binding determination, similar incubations were performed in parallel in the presence of an excess of unlabelled specific ligand. Bound and free ligands were separated by rapid filtration with Perkin-Elmer GF/B 96-unifilters pre-soaked in 0.3 % polyethylenimine. The filters were washed 3 times and the retained radioactivity measured by liquid scintillation counting in a TopCount scintillation counter (Perkin Elmer). Saturation curves were analysed by nonlinear regression using Prism software (GraphPad Software, La Jolla, USA) to determine Bmax and Kd values.
GFP determination in wild-type and TM6* strains
Cells were suspended in 0.7 mL ice-cold breaking buffer (50 mM sodium phosphate buffer pH7.4, 100 mM NaCl, 5 % glycerol, 2 mM EDTA) and glass beads added at a 1:1 ratio. Cells were disrupted in a FastPrep24 for 4 cycles of speed 6.5, 30 s with 2 min incubation on ice between cycles. Cell debris and unbroken cells were removed by centrifugation at 5,000 × g, 4°C for 3 min and the supernatant clarified and recovered at 20,000 × g, 4°C for 30 min. 150 μl of supernatant was mixed with 50 μL 1 M potassium phosphate, pH 8.0 and loaded in triplicate in black Nunc MaxiSorp 96-well plates. The fluorescence was determined on a SpectraMax Gemini XS plate reader (Molecular Devices, Wokingham, UK) with excitation and emission wavelengths of 390 nm and 510 nm respectively and a 495 nm cut-off.