Protein translocation through anthrax toxin channels formed in planar lipid bilayers

The 63-kDa fragment of the protective antigen (PA) component of anthrax toxin forms a heptameric channel, (PA₆₃)₇, in acidic endosomal membranes that leads to the translocation of edema factor (EF) and lethal factor (LF) to the cytosol. It also forms a channel in planar phospholipid bilayer membranes. What role does this channel play in the translocation of EF and LF? We report that after the 263-residue N-terminal piece of LF (LF_N) binds to its receptor on the (PA₆₃) ₇ channel and its N-terminal end enters the channel at small positive voltages to block it, LF_N is translocated through the channel to the opposite side at large positive voltages, thereby unblocking it. Thus, all of the translocation machinery is contained in the (PA₆₃)₇ channel, and translocation does not require any cellular proteins. The kinetics of this translocation are S-shaped, voltage-dependent, and occur on a timescale of seconds. We suggest that the translocation process might be explained simply by electrophoresis of unfolded LF_N through the channel, but the refolding of the N-terminal half of LF_N as it emerges from the channel may also provide energy for moving the rest of the molecule through the channel.