Figure 5.

YidC’s interaction with SecY’s lateral gate is independent of SecDFYajC, but the interaction with both the lateral gate and the channel interior requires the pmf. (a) SecY(I91pBpa) was expressed in the conditional SecDFYajC-depletion strain BL325 and cells were grown either in the presence of arabinose for SecDFYajC induction or in the presence of glucose for SecDFYajC depletion. INV were isolated and the steady-state amounts of SecY, YidC, SecD and SecF were monitored by immune detection using the appropriate antibodies. (b) The INV as in (a) were used for in vitro crosslinking. When indicated, the protonophore CCCP was added at a final concentration of 100 μM. (c) In vivo crosslinking of Bl21 cells expressing either SecY without pBpa (wt) or SecY derivatives where pBpa was inserted at the indicated positions. When indicated, CCCP (100 μM final concentration) was added before UV-induced crosslinking. Experiments were repeated at least three times and representative blots are shown.