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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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Conversion of PAheptamer to an SDS-resistant form occurs in early endosomes and is COPI dependent. (A) BHK cells were incubated at 4°C, 1 h with 500 ng/ml trypsin-nicked PA (PAn; Abrami et al., 2003) and 20 ng/ml LF, transferred to a toxin-free medium (37°C,1 h) and treated with 5 U/ml trypsin,10 min 37°C, to remove surface-bound toxin. Subcellular fractionation of postnuclear supernatants (PNS) was performed to separate early (EE) from late endosomes (LE) and heavy membranes (HM). 15 μg of protein from each fraction were analyzed by Western blotting for the presence of PA and PAheptamer, LF, rab5, and rab7. (B–D) LdlF cells were grown at the permissive (34°C) or restrictive temperature (40°C) for 18 h. (B) Cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE, a PA variant with a mutated furin cleavage site, submitted to antibody cross-linking at 4°C and further incubated at 37°C for 30 min. Surface-bound toxin was removed by an acid wash (which lead to the observed background staining). Bar, 10 μm. (C) Cells were incubated with 500 ng/ml PAn, 1 h 4°C, washed and incubated at 4°C or 37°C with a toxin-free medium for 15 min. Surface toxin was removed as in A. 40 μg of PNS was analyzed by a SDS-PAGE gel followed by Western blotting against PA. (D) ldlF CHO cells were incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of total cell extracts were analyzed by Western blotting to detect LF processed MEK1 (anti–NH2-terminal antibody), total MEK1 (anti–COOH-terminal antibody), LF, ε-COP, and PA. (E–J) BHK cells were incubated with 10 μg/ml PAn for 1 h at 4°C, warmed to 37°C for either 10 min (E) or 40 min (F–J) before fixation and preparation of ultrathin sections. Sections were labeled with rabbit anti-PA followed by 10 nm protein A gold. Labeling is evident on the plasma membrane (E, PM) and in putative early endosomal structures (F), recognized by the presence of a characteristic patch of coat material (asterisks). At later times (G–J) labeling is evident within a subset of multivesicular endosomal structures with enrichment on internal vesicles (arrowheads). Bars, 200 nm.
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fig1: Conversion of PAheptamer to an SDS-resistant form occurs in early endosomes and is COPI dependent. (A) BHK cells were incubated at 4°C, 1 h with 500 ng/ml trypsin-nicked PA (PAn; Abrami et al., 2003) and 20 ng/ml LF, transferred to a toxin-free medium (37°C,1 h) and treated with 5 U/ml trypsin,10 min 37°C, to remove surface-bound toxin. Subcellular fractionation of postnuclear supernatants (PNS) was performed to separate early (EE) from late endosomes (LE) and heavy membranes (HM). 15 μg of protein from each fraction were analyzed by Western blotting for the presence of PA and PAheptamer, LF, rab5, and rab7. (B–D) LdlF cells were grown at the permissive (34°C) or restrictive temperature (40°C) for 18 h. (B) Cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE, a PA variant with a mutated furin cleavage site, submitted to antibody cross-linking at 4°C and further incubated at 37°C for 30 min. Surface-bound toxin was removed by an acid wash (which lead to the observed background staining). Bar, 10 μm. (C) Cells were incubated with 500 ng/ml PAn, 1 h 4°C, washed and incubated at 4°C or 37°C with a toxin-free medium for 15 min. Surface toxin was removed as in A. 40 μg of PNS was analyzed by a SDS-PAGE gel followed by Western blotting against PA. (D) ldlF CHO cells were incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of total cell extracts were analyzed by Western blotting to detect LF processed MEK1 (anti–NH2-terminal antibody), total MEK1 (anti–COOH-terminal antibody), LF, ε-COP, and PA. (E–J) BHK cells were incubated with 10 μg/ml PAn for 1 h at 4°C, warmed to 37°C for either 10 min (E) or 40 min (F–J) before fixation and preparation of ultrathin sections. Sections were labeled with rabbit anti-PA followed by 10 nm protein A gold. Labeling is evident on the plasma membrane (E, PM) and in putative early endosomal structures (F), recognized by the presence of a characteristic patch of coat material (asterisks). At later times (G–J) labeling is evident within a subset of multivesicular endosomal structures with enrichment on internal vesicles (arrowheads). Bars, 200 nm.

Mentions: We have previously shown that upon heptamerization, PAheptamer is internalized, transported to early endosomes, and then rapidly degraded (Abrami et al., 2003) indicating efficient transport to lysosomes and exclusion from the recycling pathway. Here, we investigated whether PAheptamer undergoes pH-induced membrane insertion in early or in late endosomes. Early and late endosomes were isolated from toxin-treated BHK cells using a well-established subcellular fractionation protocol (Aniento et al., 1993; Gruenberg, 2001). The SDS-resistant PAheptamer, which only forms after the pH-dependent conformational change, was highly enriched in early endosomes (Fig. 1 A), co-fractionating with the small GTPase rab5 (Gruenberg, 2001), indicating that membrane insertion already occurred in early endosomes. In contrast, little SDS-resistant PAheptamer was detected in late endosomes containing rab7, presumably because degradation is extremely rapid (Abrami et al., 2003). Interestingly, LF was abundant in early endosomes and clearly detectable in late endosomes (Fig. 1 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)

Conversion of PAheptamer to an SDS-resistant form occurs in early endosomes and is COPI dependent. (A) BHK cells were incubated at 4°C, 1 h with 500 ng/ml trypsin-nicked PA (PAn; Abrami et al., 2003) and 20 ng/ml LF, transferred to a toxin-free medium (37°C,1 h) and treated with 5 U/ml trypsin,10 min 37°C, to remove surface-bound toxin. Subcellular fractionation of postnuclear supernatants (PNS) was performed to separate early (EE) from late endosomes (LE) and heavy membranes (HM). 15 μg of protein from each fraction were analyzed by Western blotting for the presence of PA and PAheptamer, LF, rab5, and rab7. (B–D) LdlF cells were grown at the permissive (34°C) or restrictive temperature (40°C) for 18 h. (B) Cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE, a PA variant with a mutated furin cleavage site, submitted to antibody cross-linking at 4°C and further incubated at 37°C for 30 min. Surface-bound toxin was removed by an acid wash (which lead to the observed background staining). Bar, 10 μm. (C) Cells were incubated with 500 ng/ml PAn, 1 h 4°C, washed and incubated at 4°C or 37°C with a toxin-free medium for 15 min. Surface toxin was removed as in A. 40 μg of PNS was analyzed by a SDS-PAGE gel followed by Western blotting against PA. (D) ldlF CHO cells were incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of total cell extracts were analyzed by Western blotting to detect LF processed MEK1 (anti–NH2-terminal antibody), total MEK1 (anti–COOH-terminal antibody), LF, ε-COP, and PA. (E–J) BHK cells were incubated with 10 μg/ml PAn for 1 h at 4°C, warmed to 37°C for either 10 min (E) or 40 min (F–J) before fixation and preparation of ultrathin sections. Sections were labeled with rabbit anti-PA followed by 10 nm protein A gold. Labeling is evident on the plasma membrane (E, PM) and in putative early endosomal structures (F), recognized by the presence of a characteristic patch of coat material (asterisks). At later times (G–J) labeling is evident within a subset of multivesicular endosomal structures with enrichment on internal vesicles (arrowheads). Bars, 200 nm.
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fig1: Conversion of PAheptamer to an SDS-resistant form occurs in early endosomes and is COPI dependent. (A) BHK cells were incubated at 4°C, 1 h with 500 ng/ml trypsin-nicked PA (PAn; Abrami et al., 2003) and 20 ng/ml LF, transferred to a toxin-free medium (37°C,1 h) and treated with 5 U/ml trypsin,10 min 37°C, to remove surface-bound toxin. Subcellular fractionation of postnuclear supernatants (PNS) was performed to separate early (EE) from late endosomes (LE) and heavy membranes (HM). 15 μg of protein from each fraction were analyzed by Western blotting for the presence of PA and PAheptamer, LF, rab5, and rab7. (B–D) LdlF cells were grown at the permissive (34°C) or restrictive temperature (40°C) for 18 h. (B) Cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE, a PA variant with a mutated furin cleavage site, submitted to antibody cross-linking at 4°C and further incubated at 37°C for 30 min. Surface-bound toxin was removed by an acid wash (which lead to the observed background staining). Bar, 10 μm. (C) Cells were incubated with 500 ng/ml PAn, 1 h 4°C, washed and incubated at 4°C or 37°C with a toxin-free medium for 15 min. Surface toxin was removed as in A. 40 μg of PNS was analyzed by a SDS-PAGE gel followed by Western blotting against PA. (D) ldlF CHO cells were incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of total cell extracts were analyzed by Western blotting to detect LF processed MEK1 (anti–NH2-terminal antibody), total MEK1 (anti–COOH-terminal antibody), LF, ε-COP, and PA. (E–J) BHK cells were incubated with 10 μg/ml PAn for 1 h at 4°C, warmed to 37°C for either 10 min (E) or 40 min (F–J) before fixation and preparation of ultrathin sections. Sections were labeled with rabbit anti-PA followed by 10 nm protein A gold. Labeling is evident on the plasma membrane (E, PM) and in putative early endosomal structures (F), recognized by the presence of a characteristic patch of coat material (asterisks). At later times (G–J) labeling is evident within a subset of multivesicular endosomal structures with enrichment on internal vesicles (arrowheads). Bars, 200 nm.
Mentions: We have previously shown that upon heptamerization, PAheptamer is internalized, transported to early endosomes, and then rapidly degraded (Abrami et al., 2003) indicating efficient transport to lysosomes and exclusion from the recycling pathway. Here, we investigated whether PAheptamer undergoes pH-induced membrane insertion in early or in late endosomes. Early and late endosomes were isolated from toxin-treated BHK cells using a well-established subcellular fractionation protocol (Aniento et al., 1993; Gruenberg, 2001). The SDS-resistant PAheptamer, which only forms after the pH-dependent conformational change, was highly enriched in early endosomes (Fig. 1 A), co-fractionating with the small GTPase rab5 (Gruenberg, 2001), indicating that membrane insertion already occurred in early endosomes. In contrast, little SDS-resistant PAheptamer was detected in late endosomes containing rab7, presumably because degradation is extremely rapid (Abrami et al., 2003). Interestingly, LF was abundant in early endosomes and clearly detectable in late endosomes (Fig. 1 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