A reconstituted system for analyzing the SecY–FtsY interaction. (A) Domain structure of E. coli FtsY (top). The FtsY regions that were cross-linked to SecY are indicated by black boxes. Crystal structure of the FtsY NG domain with the membrane targeting sequence (Stjepanovic et al., 2011; PDB accession no. 2YHS; bottom). The FtsY residues within the N domain that were cross-linked to SecY are indicated by red spheres and those within the G domain by yellow spheres. (B) Crystal structure of E. coli SecYEG (Park et al., 2014; PDB accession no. 3J45). Position 357 of SecY, where pBpa was incorporated, is indicated in red and position 111, at which the fluorophore MDCC was attached, is indicated in green. (C) SecYEG(pBpa)-PLs (SecYEG(pBpa); 10 nM SecYEG) were incubated with FtsY (1.2 µM) or buffer (−). After UV treatment for activating pBpa, the sample was extracted with Na₂CO₃ for removing excess FtsY and separated by SDS-PAGE. After Western transfer, the blot was decorated with polyclonal α-FtsY antibodies. Indicated are FtsY and the SecY–FtsY cross-link products at 130 and 190 kD. Weak cross-linking products at ∼160 kD are indicated in brackets. As a control, PLs containing SecYEG without pBpa [SecYEG(wt)] were analyzed. (D) For comparison, an in vivo cross-linking assay of E. coli cells expressing SecYEG(pBpa) was performed. After UV exposure of whole cells, SecYEG(pBpa) and its cross-link products were purified and separated by SDS-PAGE. The UV-dependent cross-link products are indicated. Independently of UV exposure, FtsY co-purifies with SecY and is visible as full-length protein and N-terminally truncated derivative (FtsY-14). (E) SecYEG(pBpa)-PLs were incubated with 1.2 µM FtsY or 1.2 µM MreB and treated as described in A. After Western transfer, the blot was decorated with polyclonal α-FtsY antibodies. (F) The material described in E was decorated with polyclonal α-MreB antibodies. The asterisk indicates an unidentified protein that is nonspecifically recognized by α-MreB in the FtsY-containing sample.