Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 48, Pages 16756-16761Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0405754101
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Funding
- NIAID NIH HHS [R01 AI022021, R37 AI022021, AI 22021] Funding Source: Medline
- NIGMS NIH HHS [GM 29210, R01 GM029210, R37 GM029210] Funding Source: Medline
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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 (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 H-6-tag to the IN 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 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 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.
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