Extracellular proteins are made in the cytosol and transported out of the cell. Certain proteins travel the opposite way, from the outside to the cytosol. Most of these proteins are toxins made by bacteria and plants, but evidence is accumulating that certain growth factors can do the same. Toxins that enter the cytosol can, according to current knowledge, be roughly classified into two main groups, those that enter from endosomes and those that enter from the endoplasmic reticulum (
Diphtheria toxin, the protein responsible for the tissue damage in diphtheria, is the most famous and best studied of these toxins. Like many other toxins acting inside cells, the active form consists of two polypeptides linked by a disulfide bond. Because the toxin is synthesized by the bacterium as a single chain protein that is subsequently cleaved, the two parts are referred to as the A and the B fragments. The B fragment consists of two functional parts that form separate domains in the crystallized protein, the receptor-binding R domain and the T domain (transmembrane) that is involved in translocation of the catalytic A fragment (or C domain) to the cytosol. The A fragment is an enzyme that ADP-ribosylates elongation factor 2 in the cytosol, leading to protein synthesis inhibition and, consequently, to cell death. Upon endocytosis of the toxin-receptor complex, the low pH in the endosome triggers unfolding of the toxin molecule, leading to insertion of the T domain into the endosomal membrane and concomitant translocation of the A fragment to the cytosol. The process can be induced already at the cell surface when cells with bound toxin are exposed to low pH.
The T domain is different from the rest of the molecule in that it consists almost entirely of -helices. In this respect, it resembles certain pore-forming toxins such as colicin A and Bacillus thuringiensis
-endotoxin (
There has been much speculation as to whether the ion-conducting channel formed by the T domain also can accommodate the A fragment, which could be translocated across the membrane through a hydrophilic channel, much the same way as export proteins are translocated from the cytosol to the lumen of the endoplasmic reticulum. The toxin must be extensively unfolded before translocation (
So why does the toxin form a channel? Proteins evolve in a modular way and it is conceivable that diphtheria toxin has evolved from a toxin that once formed lethal pores in cellular membranes. The pores made by Bacillus thuringiensis -endotoxin and other toxins that are lytic to eukaryotic cells have conductances of several hundreds and even thousands of picosiemens per pore (
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Diphtheria Toxin Translocation from the Channel's Perspective |
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-helices, TH8 and TH9, linked by an acidic loop, thus forming a hairpin that is able to insert into the membrane at low pH (
In typical lipid bilayer experiments, the cis compartment to which the toxin is added has an acidic pH as in endosomes. This is necessary to induce a conformational change required for insertion. The pH in the trans compartment is neutral, mimicking the conditions in the cytosol. Working on the topology of the inserted toxin,
The channel closure could be relieved by adding Ni2+ ions, which bind to the hexahistidine tag on the trans side of the membrane, or trypsin, which cleaves the tag off. Addition to the cis side did not have this effect. Furthermore, when a cysteine residue in the vicinity of the hexahistidine tag was biotinylated, it was observed that streptavidin, which binds to biotin, interfered with channel closure when added to the trans, but not to the cis, side of the membrane. Taken together, the results indicate that when the T domain inserts into the membrane in response to low pH, its NH2-terminal region is translocated to the trans side.
The team then went on to study translocation of toxin A fragment across the membrane, the decisive step in the transport of the toxin to its target in the cytosol. They also found that when the hexahistidine tag was attached to the NH2 terminus of the A fragment (the COOH terminus of which was joined to the T domain by a disulfide bond), the channel was closed at negative voltages (
In addition to the trick with hexahistidine, Finkelstein and colleagues also applied an approach they had earlier used with great success in studying the insertion of colicin 1a in artificial membranes (
In those experiments, they placed cysteine residues at defined positions in the protein and then bound biotin covalently to the cysteines. The modified protein was allowed to interact with membranes under different voltage conditions, and then streptavidin was added to one or the other compartment. If the biotin (and therefore the cysteine residue) was on the side where streptavidin was added, the relevant part of the colicin became unable to flip across the membrane as it was unable to carry the bulky streptavidin molecule across the membrane. When streptavidin was bound to the protein loop in the trans position, the channel stayed open irrespective of the voltage, whereas when it was bound to the loop at the cis side before translocation, a positive voltage was unable to open the channel.
In the case of diphtheria toxin, two A fragment mutants were constructed, each of which contained a single free cysteine residue. This residue was reacted with biotin, and then membrane insertion was induced. In both cases, binding of streptavidin to the A fragment in the trans position prevented the rapid closure of the channel by the histidine tag (
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Topology of the Inserted T Domain |
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From the experiments mentioned above, it is clear that the insertion of the channel-forming helices somehow pulls the NH2 terminal part of the T domain and the whole A fragment across the membrane. However, the NH2 terminus of the T domain is located at a distance from the channel-forming hairpin helices. Furthermore, the NH2-terminal region is highly hydrophilic, containing 26 charged residues within an amino acids stretch of 63 residues total. The unusual ability of the T domain to translocate the A fragment across the membrane raised questions about how the T domain inserts into the membrane.
Through maleimide chemistry, an oligopeptide containing hexahistidine was attached to T domain mutants that each contained a single cysteine residue. From the gating properties of the resulting channels, it was inferred to which side of the membrane the cysteine was localized. By studying a number of such single cysteine mutants with respect to the channel gating caused by the attached hexahistidine peptide, a new model for the membrane topology of the T domain was proposed (Fig 1). In addition to the helices TH8-9, only one other helix, TH5, in the T domain assumed a transmembrane position. The most surprising result was that the entire hydrophilic and highly charged sequence was translocated to the trans side of the membrane. One could have feared that the attachment of a hexahistidine peptide to a cysteine residue that is transported to the trans side of the membrane when the channel is inserted could have prevented the channel insertion altogether, but that appears not to be a problem. The insertion model was supported by the results of experiments in which the single cysteine residues were biotinylated, and it was then studied how addition of streptavidin to the cis and trans sides of the chamber affected channel properties.
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Relevance to Translocation in Living Cells |
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All this was done in a surprisingly simple in vitro system consisting of only toxin and lipids. This excludes the possibility that other proteins such as the toxin receptor play a necessary role in the translocation. But does the phenomenon here observed reflect what is going on when toxin enters living cells? Probably the mechanisms are similar. Also, in intact cells, the toxin forms channels with properties resembling those formed in the planar lipid membranes (
The disulfide linking the A and B fragments appears to be reduced by exposure to the cytosol during translocation in living cells (
A remaining problem that could possibly be approached by the black lipid film system is the fate of the COOH-terminal receptor-binding domain of the B fragment, the R domain. When the B fragment is inserted into membranes of living cells, this domain is shielded from the action of extracellular proteases (
With all its virtues with respect to dissection of the translocation process, the in vitro system has some disadvantages as compared with studies on living cells. The bilayer system is extremely sensitive and able to detect a single channel in a membrane that is enormous in extent compared with the surface of a cell. This is probably the reason why the toxin receptor can be disposed of in this system, while in the living cell a measurable signal can only be obtained when the receptor provides up-concentration and correct positioning of the toxin on the membrane. Also, other molecules in the intact cells could play a role in making the translocation more efficient. An obvious possibility is that chaperones in the cytosol could be required for efficient translocation of the A fragment, perhaps by facilitating the refolding of the protein. Exposure to low pH induces the molten globule state of the A fragment, and it was recently found that the T domain is able to interact with proteins in this state (
Protein toxins acting inside cells have been useful tools in the elucidation of many biological processes, such as studies of G proteins, analysis of the mechanism of vesicular fusion in neurons and other cells, and in work on intracellular transport. The recent work from the Finkelstein laboratory on the membrane insertion of diphtheria toxin challenges current ideas of how proteins interact with membranes.
Submitted: 28 February 2000
Revised: 28 February 2000
Accepted: 29 February 2000
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References |
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