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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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Related in: MedlinePlus

TrR-1 function is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu under nonreducing conditions. Partially purified CTF complex, both human and yeast, were assayed for translocation activity in vitro under nonreducing conditions using translocation buffer containing 10 μM NADPH without DTT. The TrR-1 stereospecific inhibitor cis-13-retinoic acid and the inactive trans-13-retinoic acid isomer were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro under nonreducing conditions. Excess brHsp 90 was added to c13RA-treated CTF complexes as indicated, and translocation activity in vitro was assayed under nonreducing conditions.
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fig8: TrR-1 function is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu under nonreducing conditions. Partially purified CTF complex, both human and yeast, were assayed for translocation activity in vitro under nonreducing conditions using translocation buffer containing 10 μM NADPH without DTT. The TrR-1 stereospecific inhibitor cis-13-retinoic acid and the inactive trans-13-retinoic acid isomer were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro under nonreducing conditions. Excess brHsp 90 was added to c13RA-treated CTF complexes as indicated, and translocation activity in vitro was assayed under nonreducing conditions.

Mentions: Next, we examined the effect of the TrR-1 stereo-specific inhibitor cis-13-retinoic acid on C-domain translocation (Schallreuter and Wood, 1989). The addition of cis-13-retinoic acid, but not trans-13-retinoic acid, to either human or yeast CTF complex mixtures resulted in the complete inhibition of C-domain translocation in vitro under nonreducing conditions (Fig. 8). Importantly, the addition of excess brTrR-1 to cis-13-retinoic–treated complex restored C-domain translocation activity in vitro, suggesting that cis-13-retinoic acid inhibition is TrR-1 specific. Finally, when assayed under reducing conditions (20 mM DTT), cis-13-retinoic acid had no effect on C-domain translocation (unpublished data). Together, these results indicate that TrR-1 activity plays an essential role in the translocation and/or release of the C-domain from early endosomes.


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 function is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu under nonreducing conditions. Partially purified CTF complex, both human and yeast, were assayed for translocation activity in vitro under nonreducing conditions using translocation buffer containing 10 μM NADPH without DTT. The TrR-1 stereospecific inhibitor cis-13-retinoic acid and the inactive trans-13-retinoic acid isomer were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro under nonreducing conditions. Excess brHsp 90 was added to c13RA-treated CTF complexes as indicated, and translocation activity in vitro was assayed under nonreducing conditions.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: TrR-1 function is essential for mediating DAB389IL-2 C-domain translocation from the lumen of early endosomes to the external milieu under nonreducing conditions. Partially purified CTF complex, both human and yeast, were assayed for translocation activity in vitro under nonreducing conditions using translocation buffer containing 10 μM NADPH without DTT. The TrR-1 stereospecific inhibitor cis-13-retinoic acid and the inactive trans-13-retinoic acid isomer were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro under nonreducing conditions. Excess brHsp 90 was added to c13RA-treated CTF complexes as indicated, and translocation activity in vitro was assayed under nonreducing conditions.
Mentions: Next, we examined the effect of the TrR-1 stereo-specific inhibitor cis-13-retinoic acid on C-domain translocation (Schallreuter and Wood, 1989). The addition of cis-13-retinoic acid, but not trans-13-retinoic acid, to either human or yeast CTF complex mixtures resulted in the complete inhibition of C-domain translocation in vitro under nonreducing conditions (Fig. 8). Importantly, the addition of excess brTrR-1 to cis-13-retinoic–treated complex restored C-domain translocation activity in vitro, suggesting that cis-13-retinoic acid inhibition is TrR-1 specific. Finally, when assayed under reducing conditions (20 mM DTT), cis-13-retinoic acid had no effect on C-domain translocation (unpublished data). Together, these results indicate that TrR-1 activity plays an essential role in the translocation and/or release of the C-domain from early endosomes.

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