Figure 2.

Noncompetitive binding of PpiD and YidC to the lateral gate of SecY. A, SecY–PpiD interaction was monitored in BL21 cells expressing either plasmid-borne WT SecYEG (WT) or SecY(I91pBpa)EG, carrying the UV-dependent cross-linker pBpa at position 91 within TM2a of the lateral gate. When indicated, YidC was co-expressed from the same plasmid. After in vivo UV exposure, the SecY was purified via a C-terminal His-tag using metal-affinity chromatography, and the purified sample was analyzed on SDS-PAGE followed by Western blotting and immunodetection using α-PpiD antibodies. As a control, SecY was also purified from samples that were not UV-exposed. The SecY–PpiD cross-linking product and PpiD co-purifying with SecY are indicated. A UV-dependent band migrating at ∼kDa (#) was also detected and could reflect a cross-link between SecY and YfgM, because the available antibodies against PpiD also recognize YfgM. B, same material analyzed in A was analyzed with α-YidC antibodies, and the SecY–YidC cross-linking product is indicated. The UV-dependent band observed in the absence of pBpa (*) could reflect UV-induced radical formation of aromatic amino acids, but this was not further analyzed. C, same material as in A was analyzed with α-SecY antibodies, but due to the low specificity of the antibody, only the SecY–YidC cross-link is detected in addition to the SecY dimer and SecY monomer.