Journal of Biological Chemistry
Volume 275, Issue 16, 21 April 2000, Pages 11771-11777
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MEMBRANE TRANSPORT STRUCTURE FUNCTION AND BIOGENESIS
Organization of Diphtheria Toxin in Membranes: A HYDROPHOBIC PHOTOLABELING STUDY*

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Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a membrane-inserting domain (T), and a receptor-binding domain (R). It gains entry into cells by receptor-mediated endocytosis. The low pH (∼5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subsequent translocation of the C domain into the cell, where it inactivates protein synthesis ultimately leading to cell death. We have used a highly reactive hydrophobic photoactivable reagent, DAF, to identify the segments of DT that interact with the membrane at pH 5.2. This reagent readily partitions into membranes and, on photolysis, indiscriminately inserts into lipids and membrane-inserted domains of proteins. Subsequent chemical and/or enzymatic fragmentation followed by peptide sequencing allows for identification of the modified residues. Using this approach it was observed that T domain helices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfide link was found on thetrans side leaving part of the C domain on thetrans side. This domain then comes out to thecis side via a highly hydrophobic patch corresponding to residues 134–141, originally corresponding to a β-strand in the solution structure of DT. It appears that the three helices of the T domain could participate in the formation of a channel from a DT-oligomer, thus providing the transport route to the C domain after the disulfide reductase separates the two chains.

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This work was supported by a grant-in-aid from the Department of Science and Technology. The protein sequencing was carried out under a National facility for protein sequencing supported by Department of Science and Technology, and Department of Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.