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Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway.

Abrami L, Lindsay M, Parton RG, Leppla SH, van der Goot FG - J. Cell Biol. (2004)

Bottom Line: The resulting complex is then endocytosed.Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside.Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, 1 rue Michel Servet, Geneva, Switzerland 1211.

ABSTRACT
The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

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Alteration of the dynamics of late endosomal intraluminal vesicles leads to a delay in MEK1 cleavage by LF. (A) CHO cells were incubated or not for 18 h with the anti-LBPA antibody 6c4 (50 μg/ml), washed, further incubated at 4°C for 1 h with 500 ng/ml PAn and 250 ng/ml LF, and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 20 μg of PNS was analyzed by Western blotting to detect PAheptamer, the NH2 terminus of MEK1 and LF. The amount of intact MEK1 was quantified as in Fig. 2 A (n = 4). (B) HeLa cells were transfected with siRNA against ALIX, the efficiency of which was examined 78 h later by Western blotting using antibodies against ALIX. Cells were then incubated at 4°C for 1 h with 500 ng/ml PAn and 100 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium, homogenized, and 20 μg of PNS was analyzed to detect the presence of PAheptamer and the NH2 terminus of MEK1. Quantifications were performed as in A. (C) HeLa cells were treated as in B. 80 μg of PNS were centrifuged at 100,000 g for 1 h and the pellet analyzed by Western blotting for LF. (D) CHO cells were treated as in A and incubated with 500 ng/ml PAn and 500 ng/ml FP59. 20 μg of cell lysates were loaded on native or SDS-PAGE before Western blotting against EF-2. (E) ALIX was knocked down in HeLa cells as in B, incubated with 500 ng/ml PAn with 500 ng/ml FP59, and the modification of EF2 analyzed as in D.
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fig3: Alteration of the dynamics of late endosomal intraluminal vesicles leads to a delay in MEK1 cleavage by LF. (A) CHO cells were incubated or not for 18 h with the anti-LBPA antibody 6c4 (50 μg/ml), washed, further incubated at 4°C for 1 h with 500 ng/ml PAn and 250 ng/ml LF, and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 20 μg of PNS was analyzed by Western blotting to detect PAheptamer, the NH2 terminus of MEK1 and LF. The amount of intact MEK1 was quantified as in Fig. 2 A (n = 4). (B) HeLa cells were transfected with siRNA against ALIX, the efficiency of which was examined 78 h later by Western blotting using antibodies against ALIX. Cells were then incubated at 4°C for 1 h with 500 ng/ml PAn and 100 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium, homogenized, and 20 μg of PNS was analyzed to detect the presence of PAheptamer and the NH2 terminus of MEK1. Quantifications were performed as in A. (C) HeLa cells were treated as in B. 80 μg of PNS were centrifuged at 100,000 g for 1 h and the pellet analyzed by Western blotting for LF. (D) CHO cells were treated as in A and incubated with 500 ng/ml PAn and 500 ng/ml FP59. 20 μg of cell lysates were loaded on native or SDS-PAGE before Western blotting against EF-2. (E) ALIX was knocked down in HeLa cells as in B, incubated with 500 ng/ml PAn with 500 ng/ml FP59, and the modification of EF2 analyzed as in D.

Mentions: Sorting into and formation of intraluminal vesicles occurs in early endosomes and seems to be, at that stage, a one-way street (Katzmann et al., 2002; Gruenberg and Stenmark, 2004). Once these intraluminal vesicles have reached late endosomes, some apparently acquire the ability to undergo regulated back fusion with the limiting membrane. The membrane of intraluminal vesicles indeed not only contains proteins destined to be degraded but also proteins in transit to other destinations in the cell (Kobayashi et al., 2000; Chow et al., 2002), which must get back to the limiting membrane from which budding of outgoing vesicles occurs (Gruenberg, 2001; Murk et al., 2003). To test whether this localized ability of back fusion of intraluminal vesicles could be used by LF to reach the cytoplasm, we affected one of the abundant and important components of intraluminal vesicles, the unconventional lipid lysobisphosphatidic acid (LBPA; Gruenberg, 2001). This lipid is unique to late endosomes and it was shown that feeding cells with a monoclonal antibody against LBPA, 6c4, impairs sorting of proteins and lipids leading to a traffic jam in the compartment (Kobayashi et al., 1999). We found that incubating cells with the 6c4 antibody did not affect the kinetics of formation of SDS-resistant PAheptamer as expected, but significantly delayed cleavage of MEK1 by LF (Fig. 3 A).


Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway.

Abrami L, Lindsay M, Parton RG, Leppla SH, van der Goot FG - J. Cell Biol. (2004)

Alteration of the dynamics of late endosomal intraluminal vesicles leads to a delay in MEK1 cleavage by LF. (A) CHO cells were incubated or not for 18 h with the anti-LBPA antibody 6c4 (50 μg/ml), washed, further incubated at 4°C for 1 h with 500 ng/ml PAn and 250 ng/ml LF, and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 20 μg of PNS was analyzed by Western blotting to detect PAheptamer, the NH2 terminus of MEK1 and LF. The amount of intact MEK1 was quantified as in Fig. 2 A (n = 4). (B) HeLa cells were transfected with siRNA against ALIX, the efficiency of which was examined 78 h later by Western blotting using antibodies against ALIX. Cells were then incubated at 4°C for 1 h with 500 ng/ml PAn and 100 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium, homogenized, and 20 μg of PNS was analyzed to detect the presence of PAheptamer and the NH2 terminus of MEK1. Quantifications were performed as in A. (C) HeLa cells were treated as in B. 80 μg of PNS were centrifuged at 100,000 g for 1 h and the pellet analyzed by Western blotting for LF. (D) CHO cells were treated as in A and incubated with 500 ng/ml PAn and 500 ng/ml FP59. 20 μg of cell lysates were loaded on native or SDS-PAGE before Western blotting against EF-2. (E) ALIX was knocked down in HeLa cells as in B, incubated with 500 ng/ml PAn with 500 ng/ml FP59, and the modification of EF2 analyzed as in D.
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fig3: Alteration of the dynamics of late endosomal intraluminal vesicles leads to a delay in MEK1 cleavage by LF. (A) CHO cells were incubated or not for 18 h with the anti-LBPA antibody 6c4 (50 μg/ml), washed, further incubated at 4°C for 1 h with 500 ng/ml PAn and 250 ng/ml LF, and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 20 μg of PNS was analyzed by Western blotting to detect PAheptamer, the NH2 terminus of MEK1 and LF. The amount of intact MEK1 was quantified as in Fig. 2 A (n = 4). (B) HeLa cells were transfected with siRNA against ALIX, the efficiency of which was examined 78 h later by Western blotting using antibodies against ALIX. Cells were then incubated at 4°C for 1 h with 500 ng/ml PAn and 100 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium, homogenized, and 20 μg of PNS was analyzed to detect the presence of PAheptamer and the NH2 terminus of MEK1. Quantifications were performed as in A. (C) HeLa cells were treated as in B. 80 μg of PNS were centrifuged at 100,000 g for 1 h and the pellet analyzed by Western blotting for LF. (D) CHO cells were treated as in A and incubated with 500 ng/ml PAn and 500 ng/ml FP59. 20 μg of cell lysates were loaded on native or SDS-PAGE before Western blotting against EF-2. (E) ALIX was knocked down in HeLa cells as in B, incubated with 500 ng/ml PAn with 500 ng/ml FP59, and the modification of EF2 analyzed as in D.
Mentions: Sorting into and formation of intraluminal vesicles occurs in early endosomes and seems to be, at that stage, a one-way street (Katzmann et al., 2002; Gruenberg and Stenmark, 2004). Once these intraluminal vesicles have reached late endosomes, some apparently acquire the ability to undergo regulated back fusion with the limiting membrane. The membrane of intraluminal vesicles indeed not only contains proteins destined to be degraded but also proteins in transit to other destinations in the cell (Kobayashi et al., 2000; Chow et al., 2002), which must get back to the limiting membrane from which budding of outgoing vesicles occurs (Gruenberg, 2001; Murk et al., 2003). To test whether this localized ability of back fusion of intraluminal vesicles could be used by LF to reach the cytoplasm, we affected one of the abundant and important components of intraluminal vesicles, the unconventional lipid lysobisphosphatidic acid (LBPA; Gruenberg, 2001). This lipid is unique to late endosomes and it was shown that feeding cells with a monoclonal antibody against LBPA, 6c4, impairs sorting of proteins and lipids leading to a traffic jam in the compartment (Kobayashi et al., 1999). We found that incubating cells with the 6c4 antibody did not affect the kinetics of formation of SDS-resistant PAheptamer as expected, but significantly delayed cleavage of MEK1 by LF (Fig. 3 A).

Bottom Line: The resulting complex is then endocytosed.Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside.Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, 1 rue Michel Servet, Geneva, Switzerland 1211.

ABSTRACT
The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

Show MeSH
Related in: MedlinePlus