Incorporation of a polypeptide segment into the beta-domain pore during the assembly of a bacterial autotransporter.
Bacterial autotransporters consist of an N-terminal 'passenger domain' that is transported into the extracellular space by an unknown mechanism and a C-terminal 'beta-domain' that forms a beta-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 beta-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 beta-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 beta-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.
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0538, USA.
Raffaele
R
Ieva
Kristen M
KM
Skillman
Harris D
HD
Bernstein
D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D019453:Escherichia coli O157 / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids; D018272:Porins / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D010455:Peptides; D018272:Porins; C107189:EspP protein, E coli; D012697:Serine Endopeptidases