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The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex.

Ratts R, Zeng H, Berg EA, Blue C, McComb ME, Costello CE, vanderSpek JC, Murphy JR - J. Cell Biol. (2003)

Bottom Line: The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast.In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain.Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. ratts@bu.edu

ABSTRACT
In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

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Partial purification of cytosolic proteins required to mediate DT C-domain translocation from the lumen of early endosomes in vitro. (A) Early endosomes from human T cells (HUT102/6TG), preloaded with DAB389IL-2, were incubated for 30 min at 37°C with 2 mM ATP (A) and/or 4 μg HUT102/6TG crude cytosol (C). C* denotes heat inactivation of cytosol before incubation with endosomes. Endosomes were pelleted, and the supernatant fraction (S) was assayed for DT C-domain ADP-ribosyltransferase activity by measuring the incorporation of [32P]NAD+ into AD[32P]ribosyl-EF-2 after 7% SDS-PAGE and autoradiography. (B) Time course for translocation of ADP-ribosyltransferase activity from DA189(VSV-G)B389IL-2 across the early endosomal membrane. Translocations were performed as described above, except the reactions were incubated at different temperatures (0, 15, and 37°C) for 15, 30, and 45 min. Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry. The sum of densitometry units from each pair of supernatant fluid and pellet fractions is plotted as the percentage of activity in the supernatant fractions at that time point. (n = 3; error bar denotes SD).
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fig1: Partial purification of cytosolic proteins required to mediate DT C-domain translocation from the lumen of early endosomes in vitro. (A) Early endosomes from human T cells (HUT102/6TG), preloaded with DAB389IL-2, were incubated for 30 min at 37°C with 2 mM ATP (A) and/or 4 μg HUT102/6TG crude cytosol (C). C* denotes heat inactivation of cytosol before incubation with endosomes. Endosomes were pelleted, and the supernatant fraction (S) was assayed for DT C-domain ADP-ribosyltransferase activity by measuring the incorporation of [32P]NAD+ into AD[32P]ribosyl-EF-2 after 7% SDS-PAGE and autoradiography. (B) Time course for translocation of ADP-ribosyltransferase activity from DA189(VSV-G)B389IL-2 across the early endosomal membrane. Translocations were performed as described above, except the reactions were incubated at different temperatures (0, 15, and 37°C) for 15, 30, and 45 min. Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry. The sum of densitometry units from each pair of supernatant fluid and pellet fractions is plotted as the percentage of activity in the supernatant fractions at that time point. (n = 3; error bar denotes SD).

Mentions: As shown in Fig. 1 A, on dilution of bafilomycin A1 and the addition of both ATP and cytosolic extracts to the reaction mixture, the C-domain is translocated across the endosomal membrane and released into the external medium. Moreover, preboiling the cytosolic extracts before their addition to the reaction mixture abolishes C-domain translocation. These results suggest that the C-domain translocation across the membrane of early endosomes requires cytosolic protein(s). The time course of C-domain translocation was examined using the epitope-labeled fusion protein toxin DAB189(VSV-G)B389IL-2. The cytotoxic potency of the epitope-tagged fusion toxin is almost identical to that of DAB389IL-2 (IC50 = 3 × 10−11 M vs. 4 × 10−12 M). As shown in Fig. 1 B, the ADP-ribosyltransferase activity as measured by densitometry of the combined 32P-labeled EF-2 from each paired pellet and supernatant fluid fraction is plotted as percentage of ADP-ribosyltransferase activity in the supernatant fluid. As can be seen, translocation of the C-domain is linear for up to 45 min, at which time ∼80% of the total activity is found in the supernatant fluid fraction. As reported by Lemichez et al. (1997), whereas ADP-ribosyltransferase activity was translocated to the external medium, cointernalized HRP activity was found to remain in the pellet fraction throughout the incubation period (unpublished data). These results strongly suggest that C-domain translocation is specific, and not the result of spontaneous endosomal lysis during the incubation period. Finally, in the presence of added ATP and cytosolic extracts, the translocation of the C-domain is dependent on membrane fluidity and does not occur at temperatures below 15°C.


The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex.

Ratts R, Zeng H, Berg EA, Blue C, McComb ME, Costello CE, vanderSpek JC, Murphy JR - J. Cell Biol. (2003)

Partial purification of cytosolic proteins required to mediate DT C-domain translocation from the lumen of early endosomes in vitro. (A) Early endosomes from human T cells (HUT102/6TG), preloaded with DAB389IL-2, were incubated for 30 min at 37°C with 2 mM ATP (A) and/or 4 μg HUT102/6TG crude cytosol (C). C* denotes heat inactivation of cytosol before incubation with endosomes. Endosomes were pelleted, and the supernatant fraction (S) was assayed for DT C-domain ADP-ribosyltransferase activity by measuring the incorporation of [32P]NAD+ into AD[32P]ribosyl-EF-2 after 7% SDS-PAGE and autoradiography. (B) Time course for translocation of ADP-ribosyltransferase activity from DA189(VSV-G)B389IL-2 across the early endosomal membrane. Translocations were performed as described above, except the reactions were incubated at different temperatures (0, 15, and 37°C) for 15, 30, and 45 min. Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry. The sum of densitometry units from each pair of supernatant fluid and pellet fractions is plotted as the percentage of activity in the supernatant fractions at that time point. (n = 3; error bar denotes SD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172777&req=5

fig1: Partial purification of cytosolic proteins required to mediate DT C-domain translocation from the lumen of early endosomes in vitro. (A) Early endosomes from human T cells (HUT102/6TG), preloaded with DAB389IL-2, were incubated for 30 min at 37°C with 2 mM ATP (A) and/or 4 μg HUT102/6TG crude cytosol (C). C* denotes heat inactivation of cytosol before incubation with endosomes. Endosomes were pelleted, and the supernatant fraction (S) was assayed for DT C-domain ADP-ribosyltransferase activity by measuring the incorporation of [32P]NAD+ into AD[32P]ribosyl-EF-2 after 7% SDS-PAGE and autoradiography. (B) Time course for translocation of ADP-ribosyltransferase activity from DA189(VSV-G)B389IL-2 across the early endosomal membrane. Translocations were performed as described above, except the reactions were incubated at different temperatures (0, 15, and 37°C) for 15, 30, and 45 min. Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry. The sum of densitometry units from each pair of supernatant fluid and pellet fractions is plotted as the percentage of activity in the supernatant fractions at that time point. (n = 3; error bar denotes SD).
Mentions: As shown in Fig. 1 A, on dilution of bafilomycin A1 and the addition of both ATP and cytosolic extracts to the reaction mixture, the C-domain is translocated across the endosomal membrane and released into the external medium. Moreover, preboiling the cytosolic extracts before their addition to the reaction mixture abolishes C-domain translocation. These results suggest that the C-domain translocation across the membrane of early endosomes requires cytosolic protein(s). The time course of C-domain translocation was examined using the epitope-labeled fusion protein toxin DAB189(VSV-G)B389IL-2. The cytotoxic potency of the epitope-tagged fusion toxin is almost identical to that of DAB389IL-2 (IC50 = 3 × 10−11 M vs. 4 × 10−12 M). As shown in Fig. 1 B, the ADP-ribosyltransferase activity as measured by densitometry of the combined 32P-labeled EF-2 from each paired pellet and supernatant fluid fraction is plotted as percentage of ADP-ribosyltransferase activity in the supernatant fluid. As can be seen, translocation of the C-domain is linear for up to 45 min, at which time ∼80% of the total activity is found in the supernatant fluid fraction. As reported by Lemichez et al. (1997), whereas ADP-ribosyltransferase activity was translocated to the external medium, cointernalized HRP activity was found to remain in the pellet fraction throughout the incubation period (unpublished data). These results strongly suggest that C-domain translocation is specific, and not the result of spontaneous endosomal lysis during the incubation period. Finally, in the presence of added ATP and cytosolic extracts, the translocation of the C-domain is dependent on membrane fluidity and does not occur at temperatures below 15°C.

Bottom Line: The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast.In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain.Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. ratts@bu.edu

ABSTRACT
In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

Show MeSH
Related in: MedlinePlus