The SecY–FtsY interaction requires the presence of lipids. (A) SecYEG(pBpa)-PL or SecYEG(pBpa) in detergent solution (0.03% DDM) were incubated with different FtsY concentrations and UV activated. SecYEG(pBpa)-PLs were Na₂CO₃ treated as described in the legend to Fig. 1, and SecYEG(pBpa) in detergent was directly precipitated with TCA. Pellets after centrifugation were loaded on SDS-PAGE and further treated as in Fig. 1. FtsY-14 does not efficiently interact with membranes (Weiche et al., 2008) and is therefore not visible in the carbonate-treated sample. (B) SecYEG(pBpa)-PL were incubated with wild-type FtsY or the FtsY(R198D-K200D) mutant. The conditions for cross-linking and the detection of cross-links were as in A. (C) The SecY–FtsY interaction was monitored in both PL and nanodiscs. Treatment and conditions were identical as in Fig. 1, except that cross-linking products of SecYEG(pBpa)-nanodiscs were not carbonate extracted.