Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter.
Bacterial autotransporters are proteins that contain a small C-terminal 'beta domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPDelta1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPDelta1-851 behaves as a compact monomer and strongly suggest that the channel formed by the beta domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPDelta1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP beta domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.
Skillman
Kristen M
KM
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Barnard
Travis J
TJ
Peterson
Janine H
JH
Ghirlando
Rodolfo
R
Bernstein
Harris D
HD
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D019453:Escherichia coli O157 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C107189:EspP protein, E coli; D012697:Serine Endopeptidases