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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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The in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors. (A) Fluorescence emission of 1 ng/ml SNARF-1 70 kD dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM. (B) Purified early endosomes preloaded with the pH-sensitive SNARF-1 70 kD dextran conjugates were incubated in translocation assay buffer for 20 min at 37°C with 2 mM ATP and/or 0.1 μg/μL of Mono Q-purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a fluorescence detector (model 650S; PerkinElmer). Error bars denote SD.
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fig2: The in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors. (A) Fluorescence emission of 1 ng/ml SNARF-1 70 kD dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM. (B) Purified early endosomes preloaded with the pH-sensitive SNARF-1 70 kD dextran conjugates were incubated in translocation assay buffer for 20 min at 37°C with 2 mM ATP and/or 0.1 μg/μL of Mono Q-purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a fluorescence detector (model 650S; PerkinElmer). Error bars denote SD.

Mentions: To rule out the possibility that the crude T cell and yeast extracts contained an allosteric regulator(s) of vesicular ATPase activity rather than protein(s) that are required for C-domain translocation, early endosomes were charged with a 70-kD dextran conjugated with the pH-sensitive fluorescent dye, SNARF-1. As shown in Fig. 2 A, compared with pH 7.5, the fluorescence emission of 1 ng/ml SNARF-1 is decreased approximately fourfold at pH 4.5. As measured by the quenching of fluorescence emission of SNARF-1, in vitro acidification of the early endosomal lumen occurs on dilution of bafilomycin A1 and requires the addition of 2 mM ATP to the reaction mixture (Fig. 2 B). Moreover, the time course for the acidification of early endosomes in vitro is virtually identical after the addition of either 2 mM ATP or 2 mM ATP plus partially purified T cell CTF complex.


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)

The in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors. (A) Fluorescence emission of 1 ng/ml SNARF-1 70 kD dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM. (B) Purified early endosomes preloaded with the pH-sensitive SNARF-1 70 kD dextran conjugates were incubated in translocation assay buffer for 20 min at 37°C with 2 mM ATP and/or 0.1 μg/μL of Mono Q-purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a fluorescence detector (model 650S; PerkinElmer). Error bars denote SD.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: The in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors. (A) Fluorescence emission of 1 ng/ml SNARF-1 70 kD dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM. (B) Purified early endosomes preloaded with the pH-sensitive SNARF-1 70 kD dextran conjugates were incubated in translocation assay buffer for 20 min at 37°C with 2 mM ATP and/or 0.1 μg/μL of Mono Q-purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a fluorescence detector (model 650S; PerkinElmer). Error bars denote SD.
Mentions: To rule out the possibility that the crude T cell and yeast extracts contained an allosteric regulator(s) of vesicular ATPase activity rather than protein(s) that are required for C-domain translocation, early endosomes were charged with a 70-kD dextran conjugated with the pH-sensitive fluorescent dye, SNARF-1. As shown in Fig. 2 A, compared with pH 7.5, the fluorescence emission of 1 ng/ml SNARF-1 is decreased approximately fourfold at pH 4.5. As measured by the quenching of fluorescence emission of SNARF-1, in vitro acidification of the early endosomal lumen occurs on dilution of bafilomycin A1 and requires the addition of 2 mM ATP to the reaction mixture (Fig. 2 B). Moreover, the time course for the acidification of early endosomes in vitro is virtually identical after the addition of either 2 mM ATP or 2 mM ATP plus partially purified T cell CTF complex.

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