Description:
<jats:p>We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double‐spanning protein, into the <jats:italic>Escherichia coli</jats:italic> inner membrane. Using photo‐crosslinking, the first TM (H1) was shown to insert at a Sec‐translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec‐translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs.</jats:p>