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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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Hsp 90 is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90-specific inhibitors, geldanamycin and radicicol, were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hrHsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro.
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fig6: Hsp 90 is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90-specific inhibitors, geldanamycin and radicicol, were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hrHsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro.

Mentions: Because geldanamycin and radicicol are well-known inhibitors of Hsp 90, we next examined the effect of these two agents on C-domain translocation in vitro. These agents are known to bind to the ATPase site of the chaperone and block ATP hydrolysis, thereby inhibiting refolding and release of substrate (Grenert et al., 1997; Schulte et al., 1998). Fig. 6 shows that neither the addition of geldanamycin nor radicicol alone was capable of inhibiting C-domain translocation. However, when both inhibitors were used in combination, C-domain translocation was inhibited. There are several reports demonstrating the synergistic inhibitory effects of geldanamycin and radicicol on Hsp 90, and inhibition is thought to result from either the disruption of substrate binding or the interaction with cochaperonins (Rosenhagen et al., 2001). This phenomenon appears to be Hsp 90-specific because the addition of hrHsp 90 to geldanamycin/radicicol-treated human T cell CTF complexes restored C-domain translocation (Fig. 6). Interestingly, the addition of rhHsp 90 to the geldanamycin/radicicol treated CTF complex from yeast only partially restored C-domain translocation in vitro, suggesting that these agents disrupt a species-specific Hsp 82 cochaperone interaction necessary for reconstitution of translocation activity.


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)

Hsp 90 is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90-specific inhibitors, geldanamycin and radicicol, were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hrHsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Hsp 90 is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90-specific inhibitors, geldanamycin and radicicol, were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hrHsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro.
Mentions: Because geldanamycin and radicicol are well-known inhibitors of Hsp 90, we next examined the effect of these two agents on C-domain translocation in vitro. These agents are known to bind to the ATPase site of the chaperone and block ATP hydrolysis, thereby inhibiting refolding and release of substrate (Grenert et al., 1997; Schulte et al., 1998). Fig. 6 shows that neither the addition of geldanamycin nor radicicol alone was capable of inhibiting C-domain translocation. However, when both inhibitors were used in combination, C-domain translocation was inhibited. There are several reports demonstrating the synergistic inhibitory effects of geldanamycin and radicicol on Hsp 90, and inhibition is thought to result from either the disruption of substrate binding or the interaction with cochaperonins (Rosenhagen et al., 2001). This phenomenon appears to be Hsp 90-specific because the addition of hrHsp 90 to geldanamycin/radicicol-treated human T cell CTF complexes restored C-domain translocation (Fig. 6). Interestingly, the addition of rhHsp 90 to the geldanamycin/radicicol treated CTF complex from yeast only partially restored C-domain translocation in vitro, suggesting that these agents disrupt a species-specific Hsp 82 cochaperone interaction necessary for reconstitution of translocation activity.

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