[3H]Maltose uptake in proteoliposomes

Purified recombinant MalT (full-length or trypsinized) was reconstituted at a 1:100 (w/w) ratio in preformed, Triton X-100 (0.12 % [w/w])-destabilized liposomes that were prepared with E. coli polar lipid extract (Avanti, Inc.) in 100 mM potassium phosphate, pH 7.5 and 2 mM β-mercaptoethanol. Prior to the uptake measurements, frozen proteoliposomes were subjected three times to a freeze-thaw cycle followed by 2 × 10 s sonication in a Bioruptor^®. The uptake reaction was initiated by the 20-fold dilution of the proteoliposome suspension (4.5 mg lipid/mL) into assay buffer composed of 100 mM potassium phosphate, pH 7.5 plus the 20 mM of [³H]maltose (1 Ci/mmol) at 23 °C. Reactions were stopped by the addition of ice-cold 100 mM potassium phosphate, pH 6.0 and 100 mM LiCl and filtered through Millipore 0.22 μm nitrocellulose filters. The radioactivity retained on the filters was determined with scintillation counting. Known amounts of radioactivity were used to convert counts per minute (cpm) to mol.

In order to distinguish between actual [³H]maltose accumulation on bcMalT-containing liposomes and binding to the membrane-embedded protein in the proteoliposomes, the (proteo)liposomes were incubated in the assay buffer in the presence or absence of 5 mM LDAO for 30 min at 23 °C. This concentration of detergent was chosen based on experiments in which the protein retention of LDAO-treated proteoliposomes on 0.22 μm nitrocellulose filters equaled that of proteoliposomes that were not subjected to the LDAO treatment and internally accumulated radiotracer was released. 20 μM ³H-maltose (1 Ci/mmol) was added and the samples were incubated for another 30 min (corresponding to the steady-state level of maltose accumulation (Fig. 1D). Accumulation of ³H-maltose was measured as described above.