Proteoliposomes containing the SecY-YidC fusion construct with the 14–amino acid–long linker between SecY and YidC encoding cleavage site for the PreScission protease (LEVLFQ/GP) were sonicated for 30 min and incubated with the PreScission protease (0.5 μM) for 2 hours at 14°C. The cleaved SecYEG-YidC construct was then separated by SDS-PAGE, and the gel was transferred onto a nitrocellulose membrane (0.45-μm nitrocellulose; Amersham Protran; GE Healthcare Life Sciences, Germany). After 2-hour blocking with 3% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in tris-buffered saline (TBS) buffer [50 mM tris-HCl (pH 7.5) and 150 mM NaCl], the His10-tags of the SecYEG and YidC termini were probed with Ni-NTA–horseradish peroxidase conjugate [1:1000 dilution in TBS-T buffer (TBS buffer plus 0.1% Tween 20 from Qiagen, Hilden, Germany)] overnight at 4°C. The proteins were then visualized with the Amersham ECL Western blotting detection reagents (GE Healthcare Life Sciences, MA, USA) according to the supplier’s protocol (fig. S10B). After being adsorbed to mica, proteoliposomes embedding SecYEG and YidC from the cleaved SecYEG-YidC fusion constructs were incubated a second time with the PreScission protease (2 hours at 14°C). The samples were then rinsed several times with SMFS buffer [50 mM potassium phosphate (KPi) (pH 7.2)] and used for folding experiments.

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