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Infection and Immunity, April 2006, p. 2259-2267, Vol. 74, No. 4
0019-9567/06/$08.00+0 doi:10.1128/IAI.74.4.2259-2267.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
The Cholera Toxin A13 Subdomain Is Essential for Interaction with ADP-Ribosylation Factor 6 and Full Toxic Activity but Is Not Required for Translocation from the Endoplasmic Reticulum to the Cytosol
Ken Teter,1 Michael G. Jobling,2 Danielle Sentz,2 and Randall K. Holmes2*Biomolecular Science Center and Department of Molecular Biology and Microbiology, University of Central Florida, 12722 Research Parkway, Orlando, Florida 32826,1 Department of Microbiology, Mail Stop 8333, University of Colorado School of Medicine, P.O. Box 6511, Aurora, Colorado 800452
Received 18 July 2005/ Returned for modification 4 September 2005/ Accepted 12 January 2006
Cholera toxin (CT) moves from the plasma membrane to the endoplasmic reticulum (ER) by retrograde vesicular traffic. In the ER, the catalytic CTA1 polypeptide dissociates from the rest of the toxin and enters the cytosol by a process that involves the quality control mechanism of ER-associated degradation (ERAD). The cytosolic CTA1 then ADP ribosylates Gs
, resulting in adenylate cyclase activation and intoxication of the target cell. It is hypothesized that the C-terminal A13 subdomain of CTA1 plays two crucial roles in the intoxication process: (i) it contains a hydrophobic domain that triggers the ERAD mechanism and (ii) it facilitates interaction with the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1. In this study, we examined the role(s) of the CTA13 subdomain in CT intoxication. Full-length CTA1 constructs and truncated CTA1 constructs lacking the A13 subdomain were generated and used to conduct two-hybrid studies of interactions with ARF6, in vitro enzyme assays, in vivo toxicity assays, and in vivo processing/degradation assays. Direct, plasmid-mediated expression of CTA1 constructs in the ER or cytosol of transfected CHO cells was used to perform the in vivo assays. With these methods, we found that the A13 subdomain of CTA1 is important both for interaction with ARF6 and for full expression of enzyme activity in vivo. Surprisingly, however, the A13 subdomain was not required for ERAD-mediated passage of CTA1 from the ER to the cytosol. A possible alternative trigger for CTA1 to activate the ERAD mechanism is discussed.
* Corresponding author. Mailing address: Department of Microbiology, Mail Stop 8333, University of Colorado School of Medicine, P.O. Box 6511, Aurora, CO 80045. Phone: (303) 724-4223. Fax: (303) 724-4226. E-mail: Randall.Holmes{at}UCHSC.edu.
Infection and Immunity, April 2006, p. 2259-2267, Vol. 74, No. 4
0019-9567/06/$08.00+0 doi:10.1128/IAI.74.4.2259-2267.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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