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Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

Mallard F, Antony C, Tenza D, Salamero J, Goud B, Johannes L - J. Cell Biol. (1998)

Bottom Line: This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus.B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes.Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre National de la Recherche Scientifique UMR 144, Laboratoire Mécanismes Moléculaires du Transport Intracellulaire, F-75248 Paris Cedex 05, France.

ABSTRACT
Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

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At 19.5°C, the γ-adaptin subunit of AP-1–type clathrin coats is relocalized from the TGN to EE/RE. Fluorophore-labeled B-fragment (A and C) was internalized for 1 h into HeLa  cells at 19.5°C. The cells were then either fixed directly (A and  B), or shifted for 30 min to 37°C (C and D) before fixation and  staining for γ-adaptin (B and D). Note that at 19.5°C, γ-adaptin  and B-fragment colocalized on EE/RE (A and B), and after shift  to 37°C, γ-adaptin was relocalized to the TGN, in parallel with  B-fragment accumulation in Golgi cisternae (C and D).
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Figure 8: At 19.5°C, the γ-adaptin subunit of AP-1–type clathrin coats is relocalized from the TGN to EE/RE. Fluorophore-labeled B-fragment (A and C) was internalized for 1 h into HeLa cells at 19.5°C. The cells were then either fixed directly (A and B), or shifted for 30 min to 37°C (C and D) before fixation and staining for γ-adaptin (B and D). Note that at 19.5°C, γ-adaptin and B-fragment colocalized on EE/RE (A and B), and after shift to 37°C, γ-adaptin was relocalized to the TGN, in parallel with B-fragment accumulation in Golgi cisternae (C and D).

Mentions: The BFA-induced inhibition of B-fragment exit from TfR-positive membrane compartments suggested that ADP ribosylation factor–sensitive vesicular coats could play a role in this process (Donaldson et al., 1992; Helms and Rothman, 1992). We thus set out to study the distribution of vesicular coat proteins on endosomes. Different vesicular coats have been localized on endosomes, among the best studied are coatomer type I–like coats and clathrin coats (Louvard et al., 1983; Takei et al., 1995; Whitney et al., 1995; Aniento et al., 1996; Stoorvogel et al., 1996; Daro et al., 1997; Gu et al., 1997). We here made the observation that during a 1-h incubation at 19.5°C, a component of AP-1– type clathrin coats, γ-adaptin, was shifted from its localization at steady state in the TGN to B-fragment containing EE/RE (Fig. 8, A and B). If the cells were then incubated at 37°C for 10 min (not shown) or 30 min (Fig. 8, C and D), γ-adaptin reassociated with TGN membranes while the B-fragment accumulated in the TGN/Golgi apparatus.


Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

Mallard F, Antony C, Tenza D, Salamero J, Goud B, Johannes L - J. Cell Biol. (1998)

At 19.5°C, the γ-adaptin subunit of AP-1–type clathrin coats is relocalized from the TGN to EE/RE. Fluorophore-labeled B-fragment (A and C) was internalized for 1 h into HeLa  cells at 19.5°C. The cells were then either fixed directly (A and  B), or shifted for 30 min to 37°C (C and D) before fixation and  staining for γ-adaptin (B and D). Note that at 19.5°C, γ-adaptin  and B-fragment colocalized on EE/RE (A and B), and after shift  to 37°C, γ-adaptin was relocalized to the TGN, in parallel with  B-fragment accumulation in Golgi cisternae (C and D).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132951&req=5

Figure 8: At 19.5°C, the γ-adaptin subunit of AP-1–type clathrin coats is relocalized from the TGN to EE/RE. Fluorophore-labeled B-fragment (A and C) was internalized for 1 h into HeLa cells at 19.5°C. The cells were then either fixed directly (A and B), or shifted for 30 min to 37°C (C and D) before fixation and staining for γ-adaptin (B and D). Note that at 19.5°C, γ-adaptin and B-fragment colocalized on EE/RE (A and B), and after shift to 37°C, γ-adaptin was relocalized to the TGN, in parallel with B-fragment accumulation in Golgi cisternae (C and D).
Mentions: The BFA-induced inhibition of B-fragment exit from TfR-positive membrane compartments suggested that ADP ribosylation factor–sensitive vesicular coats could play a role in this process (Donaldson et al., 1992; Helms and Rothman, 1992). We thus set out to study the distribution of vesicular coat proteins on endosomes. Different vesicular coats have been localized on endosomes, among the best studied are coatomer type I–like coats and clathrin coats (Louvard et al., 1983; Takei et al., 1995; Whitney et al., 1995; Aniento et al., 1996; Stoorvogel et al., 1996; Daro et al., 1997; Gu et al., 1997). We here made the observation that during a 1-h incubation at 19.5°C, a component of AP-1– type clathrin coats, γ-adaptin, was shifted from its localization at steady state in the TGN to B-fragment containing EE/RE (Fig. 8, A and B). If the cells were then incubated at 37°C for 10 min (not shown) or 30 min (Fig. 8, C and D), γ-adaptin reassociated with TGN membranes while the B-fragment accumulated in the TGN/Golgi apparatus.

Bottom Line: This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus.B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes.Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre National de la Recherche Scientifique UMR 144, Laboratoire Mécanismes Moléculaires du Transport Intracellulaire, F-75248 Paris Cedex 05, France.

ABSTRACT
Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

Show MeSH
Related in: MedlinePlus