Cellulose acetate filter membranes

Liposomes were prepared according to a published protocol^{42 (link)}. Purified OsSWEET2b was
reconstituted into E. coli polar lipid (Avanti) with a protein
to lipid ratio of 1:80 in KPM6.5 buffer (50 mM potassium phosphate, 2 mM
MgSO₄, pH 6.5). The proteoliposomes were extruded through 400 nm
membrane and harvested by ultracentrifugation at 100,000g for 1 hr. The
proteoliposomes were washed twice with ice-cold KPM6.5 buffer and resuspended in
the same buffer to a final concentration of 100 mg/ml immediately before the
assay. For each uptake assay, 2 μl of proteoliposomes were diluted into
100 μl of assay buffer containing 0.2 μCi of
D-[¹⁴C]glucose in the presence of 5 mM cold glucose. The reaction
mixtures were incubated for 2 hr or 3 hr at RT, and then rapidly filtered
through 0.22 μm cellulose acetate filter membranes (Millipore). The
filter membranes were immediately washed with 2 ml ice-cold reaction buffer
without any substrate and taken for liquid scintillation counting. The uptake of
fructose was performed similarly. All the experiments were repeated at least
three times independently.

The glucose transporter-deficient E. coli strain JM1100 was
obtained from Yale E.coli Genetic Stock Center. Uptake assays were
performed similarly as a published protocol^{41 (link)}. SemiSWEET wild-type (WT) and variants containing indicated point
mutations were subcloned into the pQE80 vector. The E. coli cells
containing plasmids were grown in Luria–Bertani medium at 37°C and were
induced with 50 μM IPTG for 30 min when OD₆₀₀ reached 1.5. Cells
harvested by centrifugation were washed twice with MK buffer (150mM KCl, 5mM MES pH 6.5)
before being resuspended in the MK buffer to OD₆₀₀ of 2.0. 20mM glycerol was
added to energize cells 5 min before a mixture of 2 μM D- [¹⁴C]glucose
and 1mM D-glucose was add to start the uptake reaction. After 1 min incubation with
glucose, cells were filtered onto 0.22-μm cellulose acetate filter membranes
(Millipore) under low gentle vacuum. The membranes were immediately washed with 1 ml of
ice-cold MK buffer three times and then taken for liquid scintillation counting. Control
experiments were carried out on cells containing an empty vector of pQE80. All experiments
were performed at room temperature and repeated at least three times. SemiSWEET variants
showed similar membrane expression level as the WT protein by preparing the membrane
fraction and following the same protocol as for purification of WT.