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Molecular and Cellular Biology, July 2005, p. 5492-5498, Vol. 25, No. 13
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.13.5492-5498.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Membrane Insertion by Anthrax Protective Antigen in Cultured Cells

Maen Qa'dan,¹ Kenneth A. Christensen,¹ Lei Zhang,² Thomas M. Roberts,² and R. John Collier^1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts 02115,¹ Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115²

Received 12 January 2005/ Returned for modification 9 March 2005/ Accepted 8 April 2005

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 2ß2-2ß3 loops interact to generate a 14-strand transmembrane ß-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 2ß2-2ß3 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 ß-barrel model. Besides supporting the ß-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.

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* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1930. Fax: (617) 432-0115. E-mail: jcollier{at}hms.harvard.edu.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, July 2005, p. 5492-5498, Vol. 25, No. 13
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.13.5492-5498.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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