Entrance counter-flow transport assay for human GLUTs

For the entrance counter-flow transport in proteoliposomes, the assay was started by the addition of 5 μL proteoliposomes solution (OD_600nm ~30) to 200 μL assay solution (100 mM KPi buffer at pH 7.5), containing 10 μM ¹⁴C-radiolabeled glucose (for GLUT1 wild-type or F379H mutant and GLUT2-4) or fructose (for GLUT5 wild-type or H387F mutant and GLUT2) (Moravek Biochemicals). After one minute (or different time points as specified), the transport was stopped with ice-chilled quench buffer [0.1 M KPi (pH 5.5) and 0.1 M lithium chloride]. The solution was filtered onto a cellulose nitrate membrane filter (Whatman; 0.4 μm pore size), and the filter was washed three times with quench buffer. The membrane filter was placed into a vial filled with BioSafe II scintillation liquid (Research Products International Corp.), and radioactivity was quantified with LS 6500 scintillation counter (Beckman). Compounds tested for the inhibition study were purchased from Sigma Chemicals (Supplementary Table S1). Stocks of 100 mg/ml for each compound were made in either water or dimethyl sulfoxide (DMSO). Chemicals at 1 mM final concentration in the assay volume were screened for inhibition of GLUT5 fructose transport in proteoliposomes. Tested inhibitors were added 1 min prior to addition of proteoliposomes solution. Cytochalasin B (Enzo Life Sciences) and phloretin (Alfa Aesar) were dissolved in DMSO at stock concentrations of 100 mM. Kinetic parameters were determined by nonlinear algorithm plots supplied by Prism (GraphPad Software). DMSO up to 5% (v/v) concentration in the transport assay did not affect activity. Data is presented as relative activity normalized to radioactivity of no inhibitor added as 100% and empty liposomes as 0%.