Limits...
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.

Show MeSH

Related in: MedlinePlus

The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1, and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172777&req=5

fig3: The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1, and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.

Mentions: Because C-domain translocation across the endosomal vesicle membrane requires the addition of cytosolic components to the reaction mixture, we used translocation of ADP-ribosyltransferase activity to monitor the partial purification of the active component(s) from both human T cell (HUT102/6TG) and yeast (NLY22−) extracts. After DEAE anion exchange chromatography, translocation-active fractions (150 mM–190 mM NaCl) were pooled and applied to a Sephacryl® 200 sizing column. The translocation-active fractions (250–100 kD) were pooled and further fractionated by Mono Q HPLC under conditions free of reducing agents. The translocation-active fraction was found to elute from the Mono Q column at 27.3 mS. As shown in Fig. 3 A, after fractionation on Mono Q, CTF complex activity from human T cell and yeast cell extracts was increased by 650-fold and 800-fold, respectively. Further analysis of the Mono Q-pooled fractions by SDS-PAGE and colloidal Coomassie staining revealed multiple protein bands ranging in apparent molecular mass from ∼12–100 kD (Fig. 3 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)

The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1, and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1, and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.
Mentions: Because C-domain translocation across the endosomal vesicle membrane requires the addition of cytosolic components to the reaction mixture, we used translocation of ADP-ribosyltransferase activity to monitor the partial purification of the active component(s) from both human T cell (HUT102/6TG) and yeast (NLY22−) extracts. After DEAE anion exchange chromatography, translocation-active fractions (150 mM–190 mM NaCl) were pooled and applied to a Sephacryl® 200 sizing column. The translocation-active fractions (250–100 kD) were pooled and further fractionated by Mono Q HPLC under conditions free of reducing agents. The translocation-active fraction was found to elute from the Mono Q column at 27.3 mS. As shown in Fig. 3 A, after fractionation on Mono Q, CTF complex activity from human T cell and yeast cell extracts was increased by 650-fold and 800-fold, respectively. Further analysis of the Mono Q-pooled fractions by SDS-PAGE and colloidal Coomassie staining revealed multiple protein bands ranging in apparent molecular mass from ∼12–100 kD (Fig. 3 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