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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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Efficient encounter between LF and its target occurs from late endosomes. (A) CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C (nocodazole was then present throughout the entire experiment), followed by 1 h at 4°C with 500 ng/ml PAn and 20 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed by Western blotting against PA, the NH2 terminus of MEK1 (N) and LF. The amount of intact MEK1 (A) and MKK3 (B) was quantified by densitometry and normalized to the amount at time t = 0 (n = 4, errors bars represent SDs). (C) The kinetics of decrease in intact MEK1 were compared with that of decrease in MKK3. Western blots (inset) were quantified by densitometry as in A and B. (D) RAW 264 macrophages were incubated or not with 10 μM nocodazole (2 h at 37°C), followed by 1 h at 4°C with 500 ng/ml PAn and 100 ng/ml LF. 40 μg of PNS was analyzed by Western blotting against the NH2 termini of MEK1 and MKK3. (E) HeLa cells were transfected or not with dominant-negative rab7N125I cDNA, incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed as in A (n = 4).
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fig2: Efficient encounter between LF and its target occurs from late endosomes. (A) CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C (nocodazole was then present throughout the entire experiment), followed by 1 h at 4°C with 500 ng/ml PAn and 20 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed by Western blotting against PA, the NH2 terminus of MEK1 (N) and LF. The amount of intact MEK1 (A) and MKK3 (B) was quantified by densitometry and normalized to the amount at time t = 0 (n = 4, errors bars represent SDs). (C) The kinetics of decrease in intact MEK1 were compared with that of decrease in MKK3. Western blots (inset) were quantified by densitometry as in A and B. (D) RAW 264 macrophages were incubated or not with 10 μM nocodazole (2 h at 37°C), followed by 1 h at 4°C with 500 ng/ml PAn and 100 ng/ml LF. 40 μg of PNS was analyzed by Western blotting against the NH2 termini of MEK1 and MKK3. (E) HeLa cells were transfected or not with dominant-negative rab7N125I cDNA, incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed as in A (n = 4).

Mentions: To further address whether cytoplasmic release of LF required delivery to late endosomes, we inhibited microtubule-dependent transport using the depolymerizing agent nocodazole. LF-dependent MEK1 cleavage was delayed, without affecting the formation of SDS-resistant PAheptamer (Fig. 2 A, note that degradation was inhibited as expected because access to late endosomes and lysosomes is impaired). To rule out the possibility that this delay was somehow linked to the presence of some MEK1 on late endosomes (Wunderlich et al., 2001), we also followed LF-induced cleavage of another MAPKK, MKK3, which is involved in the p38 MAPK signaling cascade, different from the MEK1-dependent ERK pathway. As for MEK1, MKK3 cleavage was delayed in nocodazole-treated cells (Fig. 2 B). Interestingly, the kinetics of cleavage of MKK3 were slower than those of MEK1 (Fig. 2 C).


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)

Efficient encounter between LF and its target occurs from late endosomes. (A) CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C (nocodazole was then present throughout the entire experiment), followed by 1 h at 4°C with 500 ng/ml PAn and 20 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed by Western blotting against PA, the NH2 terminus of MEK1 (N) and LF. The amount of intact MEK1 (A) and MKK3 (B) was quantified by densitometry and normalized to the amount at time t = 0 (n = 4, errors bars represent SDs). (C) The kinetics of decrease in intact MEK1 were compared with that of decrease in MKK3. Western blots (inset) were quantified by densitometry as in A and B. (D) RAW 264 macrophages were incubated or not with 10 μM nocodazole (2 h at 37°C), followed by 1 h at 4°C with 500 ng/ml PAn and 100 ng/ml LF. 40 μg of PNS was analyzed by Western blotting against the NH2 termini of MEK1 and MKK3. (E) HeLa cells were transfected or not with dominant-negative rab7N125I cDNA, incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed as in A (n = 4).
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fig2: Efficient encounter between LF and its target occurs from late endosomes. (A) CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C (nocodazole was then present throughout the entire experiment), followed by 1 h at 4°C with 500 ng/ml PAn and 20 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed by Western blotting against PA, the NH2 terminus of MEK1 (N) and LF. The amount of intact MEK1 (A) and MKK3 (B) was quantified by densitometry and normalized to the amount at time t = 0 (n = 4, errors bars represent SDs). (C) The kinetics of decrease in intact MEK1 were compared with that of decrease in MKK3. Western blots (inset) were quantified by densitometry as in A and B. (D) RAW 264 macrophages were incubated or not with 10 μM nocodazole (2 h at 37°C), followed by 1 h at 4°C with 500 ng/ml PAn and 100 ng/ml LF. 40 μg of PNS was analyzed by Western blotting against the NH2 termini of MEK1 and MKK3. (E) HeLa cells were transfected or not with dominant-negative rab7N125I cDNA, incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed as in A (n = 4).
Mentions: To further address whether cytoplasmic release of LF required delivery to late endosomes, we inhibited microtubule-dependent transport using the depolymerizing agent nocodazole. LF-dependent MEK1 cleavage was delayed, without affecting the formation of SDS-resistant PAheptamer (Fig. 2 A, note that degradation was inhibited as expected because access to late endosomes and lysosomes is impaired). To rule out the possibility that this delay was somehow linked to the presence of some MEK1 on late endosomes (Wunderlich et al., 2001), we also followed LF-induced cleavage of another MAPKK, MKK3, which is involved in the p38 MAPK signaling cascade, different from the MEK1-dependent ERK pathway. As for MEK1, MKK3 cleavage was delayed in nocodazole-treated cells (Fig. 2 B). Interestingly, the kinetics of cleavage of MKK3 were slower than those of MEK1 (Fig. 2 C).

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