N octyl β d glucoside β og

50 mg/mL E.coli polar lipids (Avanti Polar Lipids, Inc.) were dissolved in chloroform and methanol mixture (3:1, v/v), the organic solvent was removed by nitrogen stream. The remained lipid layer was resuspended to 20 mg/mL in assay buffer containing 40 mmol/L MES, pH 6.5, 40 mmol/L KCl, and 2 mmol/L MgSO₄. After fast frozen and thawed for 10 cycles, the mixture was extruded through 0.4 μm membrane filter (Whatman). 1% n-octyl-β-d-glucoside (β-OG, Anatrace), 20 mmol/L adenosine, and the protein hENT1 or GLUT3 (a glucose transporter) was then added into the mixture (protein: lipid = 1:100, w/w). For blank control, equal volume of the same buffer used in the final step of protein purification was added. After incubation at 4°C for 1 h, β-OG was removed by incubation with 320 mg/mL Bio-Beads SM2 (Bio-Rad) overnight. After separation from detergent-absorbed beads, the mixture was repeatedly frozen and thawed for five cycles. The proteoliposomes were extruded through 0.4 μm membrane filter again and collected by an ultracentrifugation step at 100,000 g for 1 h. The collected proteoliposomes were washed twice with the cold assay buffer. The proteoliposomes were on ice and resuspended in cold assay buffer before use with a final concentration of 100 mg/mL.

E. coli polar lipids (Avanti) were dissolved, dried, and resuspended to a final concentration of 20 mg/ml with the KPM 6.5 buffer (50 mM potassium phosphate, 2 mM MgSO₄; pH 6.5) supplemented with 50 mM D-glucose or xylose (Sigma). After freeze-and-thaw with liquid nitrogen, the liposomes were extruded through 0.4 μm polycarbonate membranes (Millipore) and incubated with 1% n-octyl-β-d-glucoside (β-OG; Anatrace) at 4 °C for 30 min. Purified protein was incubated with liposomes at a ratio of ~1:100 (w/w) for 60 min. Then β-OG was removed by incubating with Bio-Beads SM2 (Bio-Rad) overnight. The proteoliposomes were subject to freeze-and-thaw again and extruded through 0.4 μm polycarbonate membranes. The homogenized proteoliposomes were ultracentrifuged at 100,000 g at 4 °C for 1 h and rinsed twice with the ice cold KPM 6.5 buffer. Finally, the proteoliposomes were resuspended in the KPM 6.5 buffer to a final concentration of 100 mg/ml (phospholipids).