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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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Quantification of  drug effects on B-fragment  transport to the biosynthetic/ secretory pathway. (A) HeLa  cells were preincubated 2 h  with 1 μM Bafi (open  squares) and 1 μM CytoD  (open diamonds). The cells  were then transferred on ice  and incubated with 50 nM iodinated B-Glyc-KDEL for  30 min. After washing, the  cells were shifted to 37°C in  the continued presence of the  drugs. BFA (closed diamonds) was added only upon  temperature shift to 37°C.  Control cells (closed circles)  were not treated with drugs.  After the indicated times at  37°C, cells were lysed in sample buffer, and then lysates  were analyzed on 10–20%  polyacrylamide–SDS gradient gels, followed by autoradiography. The percentage of  glycosylated B-Glyc-KDEL  was expressed in function of  incubation time. The means  of three independent experiments (± SE) are shown. (B) Radiolabeled EGF was internalized into HeLa cells at 19.5°C. The cells  were then shifted to 37°C in the absence (closed circles) or presence of 1 μM Bafi (open squares), 1 μM CytoD (open circles), or  5 μg/ml BFA (open diamonds). After the indicated times at 37°C,  the number of TCA-soluble counts in the culture medium was  determined as described in Materials and Methods. A representative of two experiments is shown. (C) Iodinated B-Glyc-KDEL  was bound to HeLa cells, as described in A. The cells were then  shifted to 37°C, and 5 μg/ml BFA was added after the indicated  times (−30 min to 4 h, with respect to the beginning of B-Glyc-KDEL internalization). After 4 h at 37°C, cells were lysed and  glycosylated B-Glyc-KDEL was determined as described in A. A  representative of two experiments is shown.
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Figure 7: Quantification of drug effects on B-fragment transport to the biosynthetic/ secretory pathway. (A) HeLa cells were preincubated 2 h with 1 μM Bafi (open squares) and 1 μM CytoD (open diamonds). The cells were then transferred on ice and incubated with 50 nM iodinated B-Glyc-KDEL for 30 min. After washing, the cells were shifted to 37°C in the continued presence of the drugs. BFA (closed diamonds) was added only upon temperature shift to 37°C. Control cells (closed circles) were not treated with drugs. After the indicated times at 37°C, cells were lysed in sample buffer, and then lysates were analyzed on 10–20% polyacrylamide–SDS gradient gels, followed by autoradiography. The percentage of glycosylated B-Glyc-KDEL was expressed in function of incubation time. The means of three independent experiments (± SE) are shown. (B) Radiolabeled EGF was internalized into HeLa cells at 19.5°C. The cells were then shifted to 37°C in the absence (closed circles) or presence of 1 μM Bafi (open squares), 1 μM CytoD (open circles), or 5 μg/ml BFA (open diamonds). After the indicated times at 37°C, the number of TCA-soluble counts in the culture medium was determined as described in Materials and Methods. A representative of two experiments is shown. (C) Iodinated B-Glyc-KDEL was bound to HeLa cells, as described in A. The cells were then shifted to 37°C, and 5 μg/ml BFA was added after the indicated times (−30 min to 4 h, with respect to the beginning of B-Glyc-KDEL internalization). After 4 h at 37°C, cells were lysed and glycosylated B-Glyc-KDEL was determined as described in A. A representative of two experiments is shown.

Mentions: To obtain quantitative data on drug effects on B-fragment transport, we used a previously constructed chimeric B-fragment in which the B-fragment was fused to a N-glycosylation site and the KDEL retrieval signal, yielding a protein termed B-Glyc-KDEL (Johannes et al., 1997). As wild-type B-fragment, B-Glyc-KDEL is transported from the plasma membrane to the ER, where the chimeric protein gets progressively glycosylated. The determination of the percentage of glycosylated, cell-associated B-Glyc-KDEL at any given time point after internalization represents thus a quantitative measure for the protein's transport to the ER. Fig. 7 A shows the results of glycosylation analysis on B-Glyc-KDEL, which was incubated with HeLa cells in the presence of drugs. Control cells (Fig. 7 A, closed circles) and cells that had been pretreated for 2 h with Bafi (open squares) or CytoD (open diamonds) and that had bound iodinated B-Glyc-KDEL on ice were shifted to 37°C in the continued presence of the drugs. BFA (Fig. 7 A, closed diamonds) was only added upon temperature shift to 37°C. Cell lysates taken after the indicated times at 37°C were analyzed by autoradiography. Bafi and CytoD had no effect on B-Glyc-KDEL glycosylation (Fig. 7 A). The same results were obtained when cells were incubated in the presence of the proton pump inhibitor concanamycin B, the weak base ammonium chloride or when Bafi treatment was combined with chloroquine treatment, the weak base chloroquine assuring a rapid dissipation of endosomal pH (not shown). In contrast, no glycosylation of B-Glyc-KDEL was detectable in the presence of BFA (Fig. 7 A, closed diamonds), even after incubations of ≤15 h (not shown).


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)

Quantification of  drug effects on B-fragment  transport to the biosynthetic/ secretory pathway. (A) HeLa  cells were preincubated 2 h  with 1 μM Bafi (open  squares) and 1 μM CytoD  (open diamonds). The cells  were then transferred on ice  and incubated with 50 nM iodinated B-Glyc-KDEL for  30 min. After washing, the  cells were shifted to 37°C in  the continued presence of the  drugs. BFA (closed diamonds) was added only upon  temperature shift to 37°C.  Control cells (closed circles)  were not treated with drugs.  After the indicated times at  37°C, cells were lysed in sample buffer, and then lysates  were analyzed on 10–20%  polyacrylamide–SDS gradient gels, followed by autoradiography. The percentage of  glycosylated B-Glyc-KDEL  was expressed in function of  incubation time. The means  of three independent experiments (± SE) are shown. (B) Radiolabeled EGF was internalized into HeLa cells at 19.5°C. The cells  were then shifted to 37°C in the absence (closed circles) or presence of 1 μM Bafi (open squares), 1 μM CytoD (open circles), or  5 μg/ml BFA (open diamonds). After the indicated times at 37°C,  the number of TCA-soluble counts in the culture medium was  determined as described in Materials and Methods. A representative of two experiments is shown. (C) Iodinated B-Glyc-KDEL  was bound to HeLa cells, as described in A. The cells were then  shifted to 37°C, and 5 μg/ml BFA was added after the indicated  times (−30 min to 4 h, with respect to the beginning of B-Glyc-KDEL internalization). After 4 h at 37°C, cells were lysed and  glycosylated B-Glyc-KDEL was determined as described in A. A  representative of two experiments is shown.
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Figure 7: Quantification of drug effects on B-fragment transport to the biosynthetic/ secretory pathway. (A) HeLa cells were preincubated 2 h with 1 μM Bafi (open squares) and 1 μM CytoD (open diamonds). The cells were then transferred on ice and incubated with 50 nM iodinated B-Glyc-KDEL for 30 min. After washing, the cells were shifted to 37°C in the continued presence of the drugs. BFA (closed diamonds) was added only upon temperature shift to 37°C. Control cells (closed circles) were not treated with drugs. After the indicated times at 37°C, cells were lysed in sample buffer, and then lysates were analyzed on 10–20% polyacrylamide–SDS gradient gels, followed by autoradiography. The percentage of glycosylated B-Glyc-KDEL was expressed in function of incubation time. The means of three independent experiments (± SE) are shown. (B) Radiolabeled EGF was internalized into HeLa cells at 19.5°C. The cells were then shifted to 37°C in the absence (closed circles) or presence of 1 μM Bafi (open squares), 1 μM CytoD (open circles), or 5 μg/ml BFA (open diamonds). After the indicated times at 37°C, the number of TCA-soluble counts in the culture medium was determined as described in Materials and Methods. A representative of two experiments is shown. (C) Iodinated B-Glyc-KDEL was bound to HeLa cells, as described in A. The cells were then shifted to 37°C, and 5 μg/ml BFA was added after the indicated times (−30 min to 4 h, with respect to the beginning of B-Glyc-KDEL internalization). After 4 h at 37°C, cells were lysed and glycosylated B-Glyc-KDEL was determined as described in A. A representative of two experiments is shown.
Mentions: To obtain quantitative data on drug effects on B-fragment transport, we used a previously constructed chimeric B-fragment in which the B-fragment was fused to a N-glycosylation site and the KDEL retrieval signal, yielding a protein termed B-Glyc-KDEL (Johannes et al., 1997). As wild-type B-fragment, B-Glyc-KDEL is transported from the plasma membrane to the ER, where the chimeric protein gets progressively glycosylated. The determination of the percentage of glycosylated, cell-associated B-Glyc-KDEL at any given time point after internalization represents thus a quantitative measure for the protein's transport to the ER. Fig. 7 A shows the results of glycosylation analysis on B-Glyc-KDEL, which was incubated with HeLa cells in the presence of drugs. Control cells (Fig. 7 A, closed circles) and cells that had been pretreated for 2 h with Bafi (open squares) or CytoD (open diamonds) and that had bound iodinated B-Glyc-KDEL on ice were shifted to 37°C in the continued presence of the drugs. BFA (Fig. 7 A, closed diamonds) was only added upon temperature shift to 37°C. Cell lysates taken after the indicated times at 37°C were analyzed by autoradiography. Bafi and CytoD had no effect on B-Glyc-KDEL glycosylation (Fig. 7 A). The same results were obtained when cells were incubated in the presence of the proton pump inhibitor concanamycin B, the weak base ammonium chloride or when Bafi treatment was combined with chloroquine treatment, the weak base chloroquine assuring a rapid dissipation of endosomal pH (not shown). In contrast, no glycosylation of B-Glyc-KDEL was detectable in the presence of BFA (Fig. 7 A, closed diamonds), even after incubations of ≤15 h (not shown).

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