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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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Cytosolic delivery of DT is COPI dependent but does not require transport to late endosomes. (A and B) ldlF cells were grown at the permissive or restrictive temperature for 16 h, incubated for 7 h (A) or 2.5 h (B) at 37°C in serum-free medium with 500 ng/ml of DT or trypsin-nicked DT (DTn) or left untreated (cont.). (B) Cell lysates were prepared and the modification of EF2 analyzed as in Fig. 3 D. (C) Wild-type CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C and 15 min at 4°C, followed by 1 h at 4°C with 500 ng/ml DTn, washed and further incubated for different times at 37°C. The modification of EF2 was analyzed as above. (D) CHO cells were treated with 6c4 antibody as in Fig. 3 A and incubated with 500 ng/ml DTn and modification of EF2 analyzed. The band labeled with an asterisk is a cross-reacting protein, apparent in certain experiments. (E) ALIX was knocked down in HeLa cells as in Fig. 3 B, incubated with 500 ng/ml DTn, and modification of EF2 analyzed.
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fig4: Cytosolic delivery of DT is COPI dependent but does not require transport to late endosomes. (A and B) ldlF cells were grown at the permissive or restrictive temperature for 16 h, incubated for 7 h (A) or 2.5 h (B) at 37°C in serum-free medium with 500 ng/ml of DT or trypsin-nicked DT (DTn) or left untreated (cont.). (B) Cell lysates were prepared and the modification of EF2 analyzed as in Fig. 3 D. (C) Wild-type CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C and 15 min at 4°C, followed by 1 h at 4°C with 500 ng/ml DTn, washed and further incubated for different times at 37°C. The modification of EF2 was analyzed as above. (D) CHO cells were treated with 6c4 antibody as in Fig. 3 A and incubated with 500 ng/ml DTn and modification of EF2 analyzed. The band labeled with an asterisk is a cross-reacting protein, apparent in certain experiments. (E) ALIX was knocked down in HeLa cells as in Fig. 3 B, incubated with 500 ng/ml DTn, and modification of EF2 analyzed.

Mentions: To exclude that the various treatment had a gross effect on the endocytic pathway, we repeated the studies using a different toxin, namely diphtheria toxin (DT), known to translocate into the cytoplasm at the level of early endosomes (Papini et al., 1993; Lemichez et al., 1997). DT is also an A-B toxin, where the A subunit is, as Pseudomonas exotoxin A, an ADP-ribosyltransferase that modifies EF-2. We first tested ldlF cells at 40°C and found that they were insensitive to DT (Fig. 4 A) because ADP-ribosylation of EF-2 did not occur (Fig. 4 B). However, in contrast to what was observed for anthrax lethal toxin and PA+FP59, treatment with nocodazole had no effect on the kinetics of substrate modification by DT (Fig. 4 C), nor did incubation with the anti-LBPA antibody 6c4 (Fig. 4 D) or RNAi against ALIX (Fig. 4 E).


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)

Cytosolic delivery of DT is COPI dependent but does not require transport to late endosomes. (A and B) ldlF cells were grown at the permissive or restrictive temperature for 16 h, incubated for 7 h (A) or 2.5 h (B) at 37°C in serum-free medium with 500 ng/ml of DT or trypsin-nicked DT (DTn) or left untreated (cont.). (B) Cell lysates were prepared and the modification of EF2 analyzed as in Fig. 3 D. (C) Wild-type CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C and 15 min at 4°C, followed by 1 h at 4°C with 500 ng/ml DTn, washed and further incubated for different times at 37°C. The modification of EF2 was analyzed as above. (D) CHO cells were treated with 6c4 antibody as in Fig. 3 A and incubated with 500 ng/ml DTn and modification of EF2 analyzed. The band labeled with an asterisk is a cross-reacting protein, apparent in certain experiments. (E) ALIX was knocked down in HeLa cells as in Fig. 3 B, incubated with 500 ng/ml DTn, and modification of EF2 analyzed.
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Related In: Results  -  Collection

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fig4: Cytosolic delivery of DT is COPI dependent but does not require transport to late endosomes. (A and B) ldlF cells were grown at the permissive or restrictive temperature for 16 h, incubated for 7 h (A) or 2.5 h (B) at 37°C in serum-free medium with 500 ng/ml of DT or trypsin-nicked DT (DTn) or left untreated (cont.). (B) Cell lysates were prepared and the modification of EF2 analyzed as in Fig. 3 D. (C) Wild-type CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C and 15 min at 4°C, followed by 1 h at 4°C with 500 ng/ml DTn, washed and further incubated for different times at 37°C. The modification of EF2 was analyzed as above. (D) CHO cells were treated with 6c4 antibody as in Fig. 3 A and incubated with 500 ng/ml DTn and modification of EF2 analyzed. The band labeled with an asterisk is a cross-reacting protein, apparent in certain experiments. (E) ALIX was knocked down in HeLa cells as in Fig. 3 B, incubated with 500 ng/ml DTn, and modification of EF2 analyzed.
Mentions: To exclude that the various treatment had a gross effect on the endocytic pathway, we repeated the studies using a different toxin, namely diphtheria toxin (DT), known to translocate into the cytoplasm at the level of early endosomes (Papini et al., 1993; Lemichez et al., 1997). DT is also an A-B toxin, where the A subunit is, as Pseudomonas exotoxin A, an ADP-ribosyltransferase that modifies EF-2. We first tested ldlF cells at 40°C and found that they were insensitive to DT (Fig. 4 A) because ADP-ribosylation of EF-2 did not occur (Fig. 4 B). However, in contrast to what was observed for anthrax lethal toxin and PA+FP59, treatment with nocodazole had no effect on the kinetics of substrate modification by DT (Fig. 4 C), nor did incubation with the anti-LBPA antibody 6c4 (Fig. 4 D) or RNAi against ALIX (Fig. 4 E).

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