Syringe extruder

Labeled WT Glt_Ph and the RSMR mutant were reconstituted into liposomes, as previously described (9 (link)). Briefly, liposomes were prepared from 3:1 (w/w) mixture of E. coli polar lipid extract (Avanti Polar Lipids Inc.) and egg phosphatidylcholine in buffer A containing 50 mM Hepes/tris, pH7.4, and 200 mM KCl by extrusion through 100-nm filters (Whatman Nuclepore) using syringe extruder (Avanti Polar Lipids Inc.). Liposomes were destabilized by the addition of Triton X-100 at 1:2 (w/w) detergent-to-lipid ratio. Glt_Ph was added to destabilized liposomes at 1:1000 (w/w) PLR or at other PLRs, as indicated in the text and figures and incubated at 25°C for 30 min. Detergent was removed by six rounds of incubation with Bio-Beads (Bio-Rad) at 80 mg/ml. Liposomes were loaded with buffer A by three freeze/thaw cycles and concentrated to 25 mg/ml, ~100 μl. PEB1a variants were added to final concentrations of 0.6 μM and encapsulated by two freeze-thaw cycles. To remove excess PEB1a in the external solution, the liposomes were centrifuged for 1 hour at 49,192g at 4°C and the supernatants were discarded. Liposomes were suspended in 100 μl of buffer A and extruded through 100-nm filters 10 times.

Labeled Glt_Ph variants were reconstituted into liposomes as previously described (6 (link)). Briefly, liposomes were prepared from 3:1 (wt/wt) mixture of E. coli polar lipid extract (Avanti Polar Lipids, Inc.) and egg phosphatidylcholine in a buffer containing 50 mM Hepes/Tris, pH 7.4, and 200 mM KCl and extruded through 400-nm filters (Whatman Nuclepore) using a syringe extruder (Avanti Polar Lipids, Inc.). Liposomes were destabilized by the addition of Triton X-100 at 1:2 (wt/wt) detergent-to-lipid ratio, and Glt_Ph was added at 1:1,000 protein-to-lipid ratio. The protein/lipid mixture was incubated at 25 °C for 30 min. Detergent was removed by four to six rounds of incubation with Biobeads (Bio-Rad) at 100 mg/mL. For transport measurements, fluorescently labeled ccPEB1a-Y198F was encapsulated at a final concentration of 0.6 μM by two freeze–thaw cycles. To remove excess ccPEB1a-Y198F from the external solution, the proteoliposomes were centrifuged for 1 h at 66,125 × g at 4 °C. Resuspended proteoliposomes were concentrated to ∼25 mg/mL and extruded through 100-nm filters 11 times before imaging.