Cell-mediated Reduction and Incomplete Membrane Translocation of Diphtheria Toxin Mutants with Internal Disulfides in the A Fragment (*)
- From the Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway
Abstract
Active diphtheria toxin consists of two fragments, A and B, joined by a disulfide bond. The B fragment binds to cell surface receptors and aids in the translocation of the enzymatically active A fragment to the cytosol. Normally, the toxin A fragment enters the cytosol from acidic endosomes, but translocation can also be induced at the level of the plasma membrane by exposing cells with surface-bound toxin to low pH. Recently, we showed that disulfide bonds introduced into the A fragment by mutation are inhibitory for translocation. In the present work, we found that although the complete translocation of the A fragment is blocked, three mutant toxins underwent reduction of the interfragment disulfide bond upon low pH exposure, whereas the internal disulfide in the A fragment remained intact. In the case of two of these mutants, the A fragment was released into the extracellular medium upon exposure of cell-bound toxin to low pH. The pH profile for the release of the mutant A fragments was the same as for translocation of wild-type A fragment to the cytosol, and the release was inhibited by conditions that interfere with A fragment translocation. In the case of the third mutant, which remained cell-associated upon reduction of the interfragment disulfide bond, a translocation intermediate was detected. The results show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction takes place at an early stage in the translocation process. Our findings suggest that the translocation of the A fragment across the membrane is initiated at the C terminus.
Footnotes
-
↵* This work was supported by The Norwegian Cancer Society, The Norwegian Research Council, The Jahre Foundation, Rachel and Otto Kr. Bruuns Legacy, and The Blix Family's Fund to the Advancement of Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- NEM
-
N-ethylmaleimide
- MES
-
2-(N-morpholino)ethanesulfonic acid
- PAGE
-
polyacrylamide gel electrophoresis
- PBS
-
phosphate-buffered saline
- DTT
-
dithiotreitol
- DIDS
-
4,4′-diisothiocyanostilbene-2,2′-disulfonic acid.
-
↵2 Falnes, P. Ø., Wiedlocha, A., Rapak, A., and Olsnes, S., (1995) Biochemistry, in press.
-
↵3 Cibacron Blue inhibits anion transport and diphtheria toxin translocation in Vero cells (S. Olsnes, unpublished observations).
-
- Received April 11, 1995.
- Revision received June 5, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
