Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel.
Entry of the enzymatic components of anthrax toxin [lethal factor (LF) and edema factor] into the cytosol of mammalian cells depends on the ability of the activated protective antigen (PA63) component to form a channel (pore) in the membrane of an acidic intracellular compartment. To investigate the mechanism of translocation, we characterized N-terminally truncated forms of the PA63-binding domain of LF (LFN). Deleting 27 or 36 residues strongly inhibited acid-triggered translocation of LFN across the plasma membrane of CHO-K1 cells and ablated the protein's ability to block PA63 channels in planar lipid bilayers at a small positive voltage (+20 mV). Fusing a H6-tag to the N terminus of the truncated proteins restored both translocation and channel-blocking activities. At +20 mV, N-terminal H6 and biotin tags were accessible to Ni2+ and streptavidin, respectively, added to the trans compartment of a planar bilayer. On the basis of these findings, we propose that the N terminus of PA63-bound LF or edema factor enters the PA63-channel under the influence of acidic pH and a positive transmembrane potential and initiates translocation in an N- to C-terminal direction.
Zhang
Sen
S
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Finkelstein
Alan
A
Collier
R John
RJ
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D017931:DNA Primers; D008051:Lipid Bilayers; C030325:anthrax toxin