Nascent membrane proteins abolish SecY–FtsY cross-linking. (A) SecYEG(pBpa)-PL (10 nM final concentration SecYEG) were incubated with 1.2 µM FtsY and 50 µM GTP in the absence or presence of different concentrations of FtsQ ribosome nascent chains of 102 amino acid length (FtsQ-RNCs) or nontranslating ribosomes. After SDS-PAGE and Western transfer, the membrane was horizontally cut and the upper part was decorated with polyclonal α-FtsY antibodies (top) and the lower part with polyclonal α-uL23 antibodies (bottom). (B) Different amounts of the ribosomes and FtsQ-RNCs used in (A) were separated by SDS-PAGE and after Western transfer decorated with antibodies against Ffh, the protein component of the bacterial SRP. Purified His-tagged SRP was used as reference, and antibodies against the ribosomal protein uL2 were used to determine the ribosome concentration. A band nonspecifically recognized by α-Ffh antibodies in the FtsQ-RNC sample is marked with an asterisk. (C) SecYEG(pBpa)-PL were incubated with FtsY as in A in the presence of 50 µM GTP. When indicated, 100 nM FtsQ-RNC was added without additional SRP or with 100 nM SRP. (D) RNCs of leader peptidase (Lep-RNCs, 94 amino acid length, 10 pmol) were separated by SDS-PAGE and after Western transfer were decorated with antibodies against uL2 and Ffh. His-tagged Ffh (10 pmol) served as a control. (E) SRP-free Lep-RNCs (100 nM) were incubated with SecYEG(pBpa)-PL and FtsY as in A. When indicated, SRP (1 µM) and GTP (50 µM) were added. The top panel was decorated with antibodies against FtsY and the bottom panel with antibodies against uL23.