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.
Ieva
Raffaele
R
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0538, USA.
Skillman
Kristen M
KM
Bernstein
Harris D
HD
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