title: Two distinct steps in pullulanase secretion by Escherichia coli K12.
abstract: Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide/sec-gene-dependent general export pathway, and then inducing the pulC-O operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.
authors.affiliation: Unité de Génétique Moléculaire, Institut Pasteur, Paris, France.
authors.forename: A P
authors.initials: AP
authors.lastname: Pugsley
authors.forename: I
authors.initials: I
authors.lastname: Poquet
authors.forename: M G
authors.initials: MG
authors.lastname: Kornacker
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D004926:Escherichia coli / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006358:Hot Temperature; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013104:Spheroplasts / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D006026:Glycoside Hydrolases; C100181:pullulanase; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D004229:Dithiothreitol; D007460:Iodoacetamide