TY - JOUR
T1 - Ion conductance of the stem of the anthrax toxin channel during lethal factor translocation
AU - Schiffmiller, Aviva
AU - Finkelstein, Alan
N1 - Funding Information:
We thank Karen Jakes, Paul Kienker, Russell Thomson, and Eshwar Udho for their assistance in performing the experiments described in this paper. We also thank Myles Akabas, Karen Jakes, and Paul Kienker for their helpful comments on the manuscript, as well as Daniel Basilio for the use of his plasmids. This work was supported by National Institutes of Health research grant GM 29210 .
Publisher Copyright:
© 2014 Elsevier Ltd. All rights reserved.
PY - 2015/3/27
Y1 - 2015/3/27
N2 - The tripartite anthrax toxin consists of protective antigen, lethal factor (LF), and edema factor. PA63 (the 63-kDa, C-terminal part of protective antigen) forms heptameric channels in cell membranes that allow for the transport of LF and edema factor into the cytosol. These channels are mushroom shaped, with a ring of seven phenylalanine residues (known as the phenylalanine clamp) lining the junction between the cap and the stem. It is known that when LF is translocated through the channel, the phenylalanine clamp creates a seal that causes an essentially complete block of conduction. In order to examine ion conductance in the stem of the channel, we used Venus yellow fluorescent protein as a molecular stopper to trap LFN (the 30-kDa, 263-residue N-terminal segment of LF), as well as various truncated constructs of LFN, in mutant channels in which the phenylalanine clamp residues were mutated to alanines. Here we present evidence that ion movement occurs within the channel stem (but is stopped, of course, at the phenylalanine clamp) during protein translocation. Furthermore, we also propose that the lower region of the stem plays an important role in securing peptide chains during translocation.
AB - The tripartite anthrax toxin consists of protective antigen, lethal factor (LF), and edema factor. PA63 (the 63-kDa, C-terminal part of protective antigen) forms heptameric channels in cell membranes that allow for the transport of LF and edema factor into the cytosol. These channels are mushroom shaped, with a ring of seven phenylalanine residues (known as the phenylalanine clamp) lining the junction between the cap and the stem. It is known that when LF is translocated through the channel, the phenylalanine clamp creates a seal that causes an essentially complete block of conduction. In order to examine ion conductance in the stem of the channel, we used Venus yellow fluorescent protein as a molecular stopper to trap LFN (the 30-kDa, 263-residue N-terminal segment of LF), as well as various truncated constructs of LFN, in mutant channels in which the phenylalanine clamp residues were mutated to alanines. Here we present evidence that ion movement occurs within the channel stem (but is stopped, of course, at the phenylalanine clamp) during protein translocation. Furthermore, we also propose that the lower region of the stem plays an important role in securing peptide chains during translocation.
KW - conductance block
KW - ion-conducting channels
KW - phenylalanine clamp
KW - protein translocation
KW - single channels
UR - http://www.scopus.com/inward/record.url?scp=84924232701&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84924232701&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2014.06.016
DO - 10.1016/j.jmb.2014.06.016
M3 - Article
C2 - 24996036
AN - SCOPUS:84924232701
SN - 0022-2836
VL - 427
SP - 1211
EP - 1223
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 6
ER -