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Requirements for the Translocation of Diphtheria Toxin Fragment A across Lipid Membranes

Donovan, James J. and Simon, Melvin I. and Montal, Mauricio (1985) Requirements for the Translocation of Diphtheria Toxin Fragment A across Lipid Membranes. Journal of Biological Chemistry, 260 (15). pp. 8817-8823. ISSN 0021-9258.

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The translocation of the enzymatic moiety of diphtheria toxin, fragment A, across the membranes of pure lipid vesicles was demonstrated. A new assay, which employed vesicles made to contain radiolabeled NAD and elongation factor-2, was used to measure the appearance of the enzymatic activity of the A fragment in the vesicles. When the vesicles were exposed to a low-pH medium in the presence of diphtheria toxin, small molecules, such as NAD, escaped into the extravesicular medium, whereas large molecules mostly remained inside the vesicles. The vesicle-entrapped elongation factor-2 became ADP-ribosylated, indicating the entry of fragment A into the vesicle. The translocation of the A fragment depended upon the pH of the medium, being negligible at pH greater than 7.0 and maximal at pH 4.5. The entire toxin molecule was needed for function; neither the A fragment nor the B fragment alone was able to translocate itself across and react with the sequestered substrates. After exposure of the toxin to low pH, the entry of the A fragment was rapid, being virtually complete within 2-3 min at pH 5.5, and within 1 min at pH 4.7. Translocation occurred in the absence of any protein in the vesicle membrane. These results are consistent with the notion that the diphtheria toxin molecule enters the cytoplasm of a cell by escaping from an acidic compartment such as an endocytic vesicle.

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Additional Information:© 1985 by The American Society of Biological Chemists. Received for publication, October 26, 1983, and in revised form, April1, 1985. This investigation was supported by Research Grants EY-02084 and RR-07011 from the National Institutes of Health (to M. M.) and N00014-79-C-0798 from the Office of Naval Research (to M. M. and M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Office of Naval Research (ONR)N00014-79-C-0798
Issue or Number:15
Record Number:CaltechAUTHORS:20120628-071437492
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Official Citation:Requirements for the translocation of diphtheria toxin fragment A across lipid membranes. J J Donovan, M I Simon, and M Montal J. Biol. Chem. 1985 260: 8817-8823.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32163
Deposited By: Ruth Sustaita
Deposited On:28 Jun 2012 14:46
Last Modified:03 Oct 2019 03:58

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