The SecY–FtsY cross-links are nucleotide independent and not influenced by SRP. (A) SecYEG(pBpa)-PL (10 nM SecYEG) were incubated with FtsY (1.2 µM), either in the absence or presence of nucleotides (50 µM final concentration). Samples were processed as in Fig. 1. Indicated are FtsY and the SecY–FtsY cross-link products at 130 and 190 kD. (B) SecYEG(pBpa)-PL were incubated with FtsY as in A. When indicated SRP (1 µM) and GMP-PNP (50 µM) were added before UV exposure. (C) SecYEG(pBpa)-PL were incubated with FtsY as described in A and increasing SRP concentrations together with GMP-PNP (50 µM). Samples were further processed as in A.