Localization and assembly into the Escherichia coli envelope of a protein required for entry of colicin A.
Mutations in tolQ, previously designated fii, render cells tolerant to high concentrations of colicin A. In addition, a short deletion in the amino-terminal region of colicin A (amino acid residues 16 to 29) prevents its lethal action, although this protein can still bind the receptor and forms channels in planar lipid bilayers in vitro. These defects in translocation across the outer membrane in the tolQ cells or the colicin A mutant cannot be bypassed by osmotic shock. The TolQ protein, which is constitutively expressed at a low level, was studied in recombinant plasmid constructs allowing the expression of various TolQ fusion proteins under the control of the inducible caa promoter. The TolQ protein was thus "tagged" with an epitope from the colicin A protein for which a monoclonal antibody is available. A fusion protein containing the entire TolQ protein plus the 30 N-terminal residues of colicin A was shown to complement the tolQ mutation. Pulse-chase labeling followed by gradient fractionation indicated that the bulk of the overproduced fusion protein was rapidly incorporated into the inner membrane, with small amounts localized to regions corresponding to the attachment sites between inner and outer membranes and to the outer membrane itself. However, most of the protein was rapidly degraded, leaving only that localized to the attachment sites and the outer membrane remaining at very late times of chase.
Bourdineaud
J P
JP
Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, Marseille, France.
Howard
S P
SP
Lazdunski
C
C
D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003087:Colicins / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism
D003087:Colicins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins