LacY, YidC, SecYEG, or SecYEG-YidC fusion construct were reconstituted into liposomes formed from PE (Avanti Polar Lipids) and PG (Avanti Polar Lipids) (ratio, 3:1). LacY and YidC were reconstituted using the dilution method (25). Briefly, purified LacY or YidC was mixed with PE/PG phospholipids dissolved in 1.2% octyl glucoside (Maumee) at a lipid-to-protein ratio of 5 (w/w). The mixtures were incubated for 20 min on ice. After the incubation time, mixtures were quickly diluted 50-fold in 50 mM NaPi at pH 7.5. Centrifugation (100,000g) was applied for 1 hour to collect proteoliposomes, which were then suspended in 50 mM NaPi at pH 7.5 and flash-frozen in liquid nitrogen.

Before SecYEG or SecYEG-YidC was reconstituted, the samples were dialyzed three times for 1 hour in 20 mM tris-HCl (pH 8), 300 mM NaCl, and 0.03% DDM. Then, purified SecYEG or SecYEG-YidC was mixed with PE/PG (3:1, mol/mol) liposomes in a lipid-to-protein ratio of 10 (SecYEG) and 30 (SecYEG-YidC) (w/w) and extruded 15 times. To remove the detergent, activated Biobeads were added (twice for 1 hour at 4°C). The proteoliposomes were collected by centrifugation at 4°C (Airfuge, 10 min, 20 psi), resuspended in 50 mM NaPi (pH 8.0), and stored on ice.

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