TY - JOUR
T1 - Role of CypA and Hsp90 in membrane translocation mediated by anthrax protective antigen
AU - Dmochewitz, Lydia
AU - Lillich, Maren
AU - Kaiser, Eva
AU - Jennings, Laura D.
AU - Lang, Alexander E.
AU - Buchner, Johannes
AU - Fischer, Gunter
AU - Aktories, Klaus
AU - Collier, R. John
AU - Barth, Holger
PY - 2011/3
Y1 - 2011/3
N2 - Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LFNDTA. LFN mediates uptake of LFNDTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LFNDTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LFNDTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LFNDTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LFNDTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LFNDTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LFNDTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.
AB - Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LFNDTA. LFN mediates uptake of LFNDTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LFNDTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LFNDTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LFNDTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LFNDTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LFNDTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LFNDTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.
UR - http://www.scopus.com/inward/record.url?scp=79951470779&partnerID=8YFLogxK
U2 - 10.1111/j.1462-5822.2010.01539.x
DO - 10.1111/j.1462-5822.2010.01539.x
M3 - Article
C2 - 20946244
AN - SCOPUS:79951470779
SN - 1462-5814
VL - 13
SP - 359
EP - 373
JO - Cellular Microbiology
JF - Cellular Microbiology
IS - 3
ER -