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 (PA ₆₃) 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 PA ₆₃-binding domain of LF (LF _N). Deleting 27 or 36 residues strongly inhibited acid-triggered translocation of LF _N across the plasma membrane of CHO-K1 cells and ablated the protein's ability to block PA ₆₃ channels in planar lipid bilayers at a small positive voltage (+20 mV). Fusing a H ₆-tag to the N terminus of the truncated proteins restored both translocation and channel-blocking activities. At +20 mV, N-terminal H ₆ and biotin tags were accessible to Ni ²⁺ 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 PA ₆₃-bound LF or edema factor enters the PA ₆₃-channel under the influence of acidic pH and a positive transmembrane potential and initiates translocation in an N- to C-terminal direction.