Open AccessMembrane Insertion by Anthrax Protective Antigen in Cultured Cells
Author(s) -
Maen Qa'Dan,
Kenneth A. Christensen,
Lei Zhang,
Thomas M. Roberts,
R. John Collier
Publication year - 2005
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.25.13.5492-5498.2005
Subject(s) - biophysics , transmembrane protein , conformational change , membrane , cytosol , anthrax toxin , cysteine , biology , endosome , transmembrane domain , cell membrane , lipid bilayer fusion , biochemistry , intracellular , enzyme , fusion protein , recombinant dna , receptor , gene
The enzymatic moieties of anthrax toxin enter the cytosol of mammalian cells via a pore in the endosomal membrane formed by the protective antigen (PA) moiety. Pore formation involves an acidic pH-induced conformational rearrangement of a heptameric precursor (the prepore), in which the seven 2beta2-2beta3 loops interact to generate a 14-strand transmembrane beta-barrel. To investigate this model in vivo, we labeled PA with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) at cysteine residues introduced into the 2beta2-2beta3 loop. Each labeled PA was bound to CHO cells, and NBD fluorescence was monitored over time in stirred cell suspensions or by confocal microscopy. A strong increase was observed with NBD at positions 305, 307, 309, and 311, sites where side chains are predicted to face the bilayer, and little change was seen at residues 304, 306, 308, 310, and 312, sites where side chains are predicted to face the pore lumen. The increase at position 305 was inhibited by membrane-restricted quenchers, low temperature, or various reagents known to affect toxin action. Of the 24 NBD attachment sites examined, all but three gave results qualitatively consistent with the beta-barrel model. Besides supporting the beta-barrel model of membrane insertion, our results describe the time course of insertion and identify PA residues where NBD gives a strong signal upon membrane insertion in vivo.