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Two distinct steps in pullulanase secretion by Escherichia coli K12
Author(s) -
Pugsley A. P.,
Poquet I.,
Kornacker M. G.
Publication year - 1991
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1991.tb00760.x
Subject(s) - signal peptide , secretion , pullulanase , isopycnic , biochemistry , cytoplasm , vesicle , escherichia coli , lysis , biophysics , chemistry , biology , enzyme , membrane , centrifugation , peptide sequence , gene
Summary 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—0 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.