Incorporation of a polypeptide segment into the 3-domain pore during the assembly of a bacterial autotransporter

• Published in: Molecular microbiology Vol. 67; no. 1; p. 188
• Main Authors: Ieva, Raffaele, Skillman, Kristen M, Bernstein, Harris D
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell Publishing Ltd 01-01-2008
• Subjects: Bacteriology; E coli; Peptides; Proteases; Proteins
• ISSN: 0950-382X; 1365-2958

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. [PUBLICATION ABSTRACT]