Yeast Assay for GLUT5-Mediated Fructose Transport

GLUT5 (GLUT5tr^S72Y or GLUT5tr^S76I) expressing EBY.VW4000 yeast strains were grown in 10 ml of YEPM media with 100 µg/L antibiotic G-418, at 29 °C, in an incubator with shaking (220 rpm) for one day. Two mililiters of cell culture were centrifuged at 10,000 g for 30 seconds and the pellet was resuspended in 50 ml of YEPF media with 100 µg/L G-418. The cells were grown as above to OD_600nm ~ 10 (for 1–2 days), then harvested by centrifugation at 5,000 g for 2 minutes. The cell pellet was resuspended in 50 ml PBS buffer and centrifuged once more. The pellet was resuspended in PBS buffer at OD_600nm~1.5. Cells that did not express GLUT5 were used as background control. Transport assay was initiated at 22 °C by the addition of 2 μl of ¹⁴C-radiolabeled fructose (50 nCi, 0.2 nmol) and various concentrations of cold-fructose to 100 or 200 μl yeast cell solution with/without the inhibitor N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA, Enamine) or (−)-epicatechin-gallate (ECG, Cayman). The transport was stopped after 20 minutes with ice-chilled quench buffer (0.1 M KPi, pH 5.5, 0.1 M LiCl), the solution was filtered with 0.4 µm cellulose nitrate membrane filter (Whatman), and the filter was washed twice with the quench buffer. The membrane filter was placed into a vial filled with BioSafe II scintillation liquid (Research Products International Corp.) and radioactivity was measured with Becker LS 6500 Multi-purpose Scintillation Counter. Kinetic parameters were determined by non-linear algorithm plots supplied by Prism (GraphPad Software).