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.
|Original language||English (US)|
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Nov 30 2004|
ASJC Scopus subject areas