Jul 25, 2016 - 0 1985 by The American Society of Biological Chemists. Inc. VOl. ... Pathology, University of Florida, Gainesville, FL 32610. .... Samples were subjected to electrophoresis on 10% (w/v) sodium .... the fifth bar indicates ADP-ribosylation when the vesicles are disrupted with 1% cholate (w/v) before diphtheria.
VOl. 260. No. 15. Issue of July 25, pp. e174623 1985 Prrnted in irS.A.
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists. Inc.
Requirements for the Translocation of Diphtheria Toxin Fragment A across Lipid Membranes* (Received for publication, October 26, 1983, and in revised form, April 1,1985)
James J. Donovan$, MelvinI. SimonQ, and Mauricio Montal From the Departments of Physics and Bwbgy, University of California, San Diego, La Jolla, California92093
The translocation of the enzymatic moiety of diph- attacks elongation factor-2 (EF-2l) (7,8) by catalyzing the of pure transfer of the ADP-ribose moiety from NAD to a modified theria toxin, fragment A, across the membranes lipid vesicles was demonstrated. A new assay, which histidine residue on EF-2 (9). This inactivates the factor, employed vesicles made to contain radiolabeled NAD arresting protein synthesis. and elongation factor-2, was used to measure the apThe potency of the toxin seems to depend on its chemical pearance of the enzymatic activity of the A fragment state. Native preparations of diphtheria toxin contain a mixin the vesicles. When the vesicles were exposed to a ture of forms of the protein. The major fraction of the toxin low-pH medium in the presence of diphtheria toxin, is a single polypeptidechain with two disulfide bridges, and a small molecules, suchas NAD, escapedinto the extra- smaller amount of the toxin is “nicked” toxin, in which the vesicular medium,whereas large molecules mostlyre- peptide chain is cleaved at one point (10). The resulting two mained inside the vesicles. The vesicle-entrapped elon-fragments, A and B, remain joined by a disulfide bond (11). gation factor-2 becameADP-ribosylated,indicating the entry of fragment A into the vesicle. The translo- Intact toxin can also be converted in vitro to nicked toxin by cation of theA fragment depended upon thepH of the a gentle treatment with trypsin (10). Sandvig and Olsnes (12) medium, being negligible at pH > 7.0 and maximal at showed that nicking increases the potency of the toxin and pH 4.5. The entire toxin molecule was needed for func- they suggest that nicking is an essential step in intoxication. tion; neither theA fragment nor theB fragment alone Uchida et al. (13) showed that neither of the two fragments was able to translocate itself across and reactwith the alone is toxic, but that after separation they can be rejoined, sequestered substrates. After exposure of the toxin to recovering toxicity in the reconstituted protein. This apparlow pH, the entry of the A fragment was rapid, being ently occurs because eachof the two fragments is responsible schemejust outlined. The B fragment virtually complete within 2-3 min at pH 5.5, and for different steps in the within 1 min at pH 4.7. Translocation occurredin the contains a binding site for the cell surface receptor, and the absence ofany protein in thevesicle membrane. These A fragment contains the active site for the enzymatic reaction results are consistentwith the notion that the diphthe-with NAD and EF-2. Thus, A alone cannot bind to the cells, ria toxin molecule enters the cytoplasm of a cell by and B alone, whileit binds, cannot react with EF-2. escaping from an acidic compartment such as an enThe mechanism of toxin entry and the identity of the docytic vesicle. fragment responsible for entry are not known with certainty. Several possible mechanisms have been proposed. Boquet and co-workers (14, 15) found that diphtheria toxin contains a hydrophobic domain which they suggested acts in the entry Diphtheria toxin is a 63,000-dalton protein secreted by of A into thecytoplasm. They proposed a model in which the certain strains of Corynebacterium diphtheriae. The protein toxin enters the cell directly through the plasma membrane. is toxic to most eucaryotic cells and species, including man, In their model, the B fragment forms a pore in the plasma andthe effect of the toxin on cellsis to inhibit protein membrane, and the A fragment passes through it into the synthesis (1).The intoxication process is thought to proceed cytoplasm. They also suggested that the B fragment must be in three steps (2, 3). First, the toxin molecule binds to a clipped (at a location other than the nicking site) before this specific receptor on the surface of the cell. The natureof this occurs. A second modelwas developedin which the diphtheria receptor is a matter of controversy; it has beenvariously toxin molecule is endocytosed after binding to the receptor suggested that the receptor is a peripheral membrane protein (16). In this model, the toxin finds its way into an acidic (4), an integral membrane glycoprotein (5), or a phospholipid intracellular compartment, generally assumed to be a lyso(6). Second,the toxin is internalized and enters the cytoplasm. some or acidified endocytic vesicle, where it is triggered by Third, once in the cytoplasm, diphtheria toxin enzymatically the low pHto escape intothe cytoplasm. This model is * This investigation was supported by Research Grants EY-02084 supported by the fact that lysosomotropic drugsprotect cells and RR-07011 from the National Institutes of Health (to M. M.) and from intoxication (17, 18).More recently, Draper and Simon N00014-79-C-0798 from the Office of Naval Research (to M. M. and (19) and Sandvig and Olsnes (12,20) showed that diphtheria M. S.). The costs of publication of this article were defrayed in part toxin can be induced to enter the cytoplasm directly through by the payment of page charges. This article musttherefore be hereby the plasma membranewhen the environment on the cell marked “advertisement” in accordance with 18 U.S.C. Section 1734 exterior is changedto mimic the lysosomal environment, that solely to indicate this fact. is, when the medium bathing the cells is acidified. Thus, low National Institutes of Health postdoctoral trainee for part of this work. Present address: Department of Comparative and Experimental pH can act as a trigger, inducing fragment A to cross the Pathology, University of Florida, Gainesville, FL 32610. To whom membrane barrier and enter the cell.However, these “pH shock” studies do not address the question of the mechanism correspondence should be addressed. § Present address: Department of Biology, California Institute of Technology, Pasadena, CA 91125.
8817
The abbreviation used is: EF-2, elongation factor-2.
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Translocation of Diphtheria Toxin
of the transport of fragment A, nor do they define whether some membrane component, such as a receptor or protease, is needed fortoxin entry. Our approach to answer questions about the mechanism of toxin entry exploits the advantages of model systems where the composition of the membrane and theaqueous phases on both sides of the membrane can be controlled precisely. We showed previously(21,22) that diphtheria toxin forms channels in planar lipid bilayer membranes in the absence of any receptor protein, and that this occurs only at low pH (
