Incorporation of a polypeptide segment into the β‐domain pore during the assembly of a bacterial autotransporter

Summary Bacterial autotransporters consist of an N‐terminal ‘passenger domain’ that is transported into the extracellular space by an unknown mechanism and a C‐terminal ‘β‐domain’ that forms a β‐barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the β‐domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the β‐domain pore. By examining the accessibility of tobacco etch virus protease sites and single‐cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre‐translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP β‐domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre‐formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.