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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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TrR-1 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22− (Y) partially purified CTF complexes were depleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2′,5′ ADP-Sepharose. TrR-1–depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions. Either partially purified CTF complex or brTrR-1 was added back to TrR-1–depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of 2′,5′ ADP-Sepharose affinity-purified yeast TrR-1 (AP), marker (M). Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis.
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fig7: TrR-1 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22− (Y) partially purified CTF complexes were depleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2′,5′ ADP-Sepharose. TrR-1–depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions. Either partially purified CTF complex or brTrR-1 was added back to TrR-1–depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of 2′,5′ ADP-Sepharose affinity-purified yeast TrR-1 (AP), marker (M). Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis.

Mentions: Because TrR-1 was also identified by MS sequence analysis of CTF complexes from human T cell and yeast extracts, we have used both immunoprecipitation and specific inhibitors to demonstrate a functional role of TrR-1 in the translocation and/or release of the C-domain from early endosomes. As shown in Fig. 7 A, immunoprecipitation of TrR-1 from human CTF complexes and 2′,5′-ADP-Sepharose affinity chromatographic depletion of yeast TrR-1 from CTF complex mixtures abolished C-domain translocation in vitro. Because there was no significant loss of ADP-ribosyltransferase activity in the pellet fractions (unpublished data), we conclude that there is no pool of translocated but nonrefolded DT C-domain. Reconstitution experiments in which bovine recombinant (br) TrR-1 was added back to TrR-1-depleted CTF complexes, from both T cell and yeast, failed to restore C-domain translocation (unpublished data). Because these experiments were performed under conditions known to reduce the interchain disulfide bond between the C- and T-domains, these results suggest that TrR-1 is a component of a complex and that another factor(s) essential for translocation was codepleted with TrR-1 (Fig. 7 B).


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)

TrR-1 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22− (Y) partially purified CTF complexes were depleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2′,5′ ADP-Sepharose. TrR-1–depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions. Either partially purified CTF complex or brTrR-1 was added back to TrR-1–depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of 2′,5′ ADP-Sepharose affinity-purified yeast TrR-1 (AP), marker (M). Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172777&req=5

fig7: TrR-1 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22− (Y) partially purified CTF complexes were depleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2′,5′ ADP-Sepharose. TrR-1–depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions. Either partially purified CTF complex or brTrR-1 was added back to TrR-1–depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of 2′,5′ ADP-Sepharose affinity-purified yeast TrR-1 (AP), marker (M). Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis.
Mentions: Because TrR-1 was also identified by MS sequence analysis of CTF complexes from human T cell and yeast extracts, we have used both immunoprecipitation and specific inhibitors to demonstrate a functional role of TrR-1 in the translocation and/or release of the C-domain from early endosomes. As shown in Fig. 7 A, immunoprecipitation of TrR-1 from human CTF complexes and 2′,5′-ADP-Sepharose affinity chromatographic depletion of yeast TrR-1 from CTF complex mixtures abolished C-domain translocation in vitro. Because there was no significant loss of ADP-ribosyltransferase activity in the pellet fractions (unpublished data), we conclude that there is no pool of translocated but nonrefolded DT C-domain. Reconstitution experiments in which bovine recombinant (br) TrR-1 was added back to TrR-1-depleted CTF complexes, from both T cell and yeast, failed to restore C-domain translocation (unpublished data). Because these experiments were performed under conditions known to reduce the interchain disulfide bond between the C- and T-domains, these results suggest that TrR-1 is a component of a complex and that another factor(s) essential for translocation was codepleted with TrR-1 (Fig. 7 B).

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