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Pathways Followed By Protein Toxins into Cells

Posted on: Thursday, 17 June 2004, 06:00 CDT

Abstract

A number of protein toxins have an enzymatically active part, which is able to modify a cytosolic target. Some of these toxins, for instance ricin, Shiga toxin and cholera toxin, which we will focus on in this article, exert their effect on cells by first binding to the cell surface, then they are endocytosed, and subsequently they are transported retrogradely all the way to the ER before translocation of the enzymatically active part to the cytosol. Thus, studies of these toxins can provide information about pathways of intracellular transport. Retrograde transport to the Golgi and the ER seems to be dependent not only on different Rab and SNARE proteins, but also on cytosolic calcium, phosphatidylinositol 3-kinase and cholesterol. Comparison of the three toxins reveals differences indicating the presence of more than one pathway between early endosomes and the Golgi apparatus or, alternatively, that transport of different toxin-receptor complexes present in a certain subcompartment is differentially regulated.

Key words: Toxin - endocytosis - Golgi apparatus - lipids - ricin

Introduction

The two bacterial toxins Shiga toxin and cholera toxin and the plant toxin ricin (Figure 1) are representatives of the group of protein toxins which have one moiety of the molecule (the B-moiety) that binds to a cell-surface receptor and another moiety (the A- moiety) which enters the cytosol and exerts the effect of the toxin (for review, see (Sandvig and van Deurs, 2002; Schiavo and van der Goot, 2001)). Since the A-moieties are enzymes, only a few toxin molecules are sufficient to have drastic effects on the cells. In some cases, like for Shiga toxin and ricin, where the A-moieties inactivate ribosomes, the toxins will very efficiently kill the cells. In the case of cholera toxin the A-moiety is able to ADP- ribosylate the [alpha] subunit of heterotrimeric G^sub s^ proteins thereby constitutively activating adenylyl cyclase and increasing the level of cAMP. For other toxins the targets are different (Schiavo and van der Goot, 2001).

There are a number of good reasons to investigate the mechanisms of action of these toxins. Several of them are still a problem in connection with infectious diseases. Not only is Shiga toxin secreted by Shigella dysenteriae, but Escherichia coli able to secrete Shiga-like toxins seems to be an increasing problem (Paton and Paton, 1998; Andreoli et al., 2002). Food poisoning caused by such bacteria is associated with diarrhea, and the secreted toxins can also give kidney failure, especially in children. Since the toxins so efficiently can kill cells, several investigators are using them in targeted cell killing (Gura, 2002; Kreitman, 2001; Frankel et al., 2000; Sandvig and van Deurs, 2002). The toxins or their enzymatically active parts are for instance coupled to antibodies against cancer cells. There is now promising results from clinical trials (Kreitman, 2003). Another approach in medicine is to use the toxins for delivery of epitopes into cells with the consequence that the epitope is presented at the cell surface by MHC class I (Sandvig and van Deurs, 2002; Noakes et al., 1999; Haicheur et al., 2000; Lee et al., 1998). In principle this works, epitopes added to different toxins are in fact presented by MHC class I, although it is not clear in all cases whether the epitope is in fact released into the cytosol and then transported into the ER by the TAP transporter. The possibility exists that the epitope is released in another cellular compartment. The toxins have also proven valuable as tools in cell biology. One can use them to study cellular processes and transport routes (Sandvig and van Deurs, 2002), and there is no doubt that new knowledge has been obtained about endocytosis and intracellular transport from investigations of toxin entry into cells.

Fig. 1. Crystallographic and schematic structures of ricin, Shiga toxin and cholera toxin. Ricin: PDB protein data bank: 2AA1; Shiga toxin: PDB protein data bank: 1DMO; Cholera toxin: protein data bank: 1XTC. Below the Crystallographic structures the corresponding schematic ones are shown. As indicated, the A fragment of Shiga toxin is cleaved by the target cell, whereas cholera toxin is cleaved by the bacteria that produce the toxin.

Studies of toxin transport - methodological considerations

Measurements of the toxic end effects, i.e. inhibition of protein synthesis in the case of ricin or Shiga toxin, or quantification of the increase in cAMP levels after treatment with cholera toxin, normally constitute sensitive test systems for arrival of the enzymatically active subunits in the cytosol. However, in some cases such test systems cannot be used to estimate the amounts of toxin transferred to the cytosol. Addition of drugs or change in conditions might affect the cell such that cytosolic degradation of the toxin is changed, giving a wrong estimate. Furthermore, in the case of cholera toxin where the enzymatically active part of the toxin after entry into the cytosol modifies membrane-associated G^sub s^ proteins which again need to interact with the membrane- associated adenylyl cyclase to increase the level of cAMP, addition of membrane-active agents might of course not only modify uptake of cholera toxin into the cells by endocytosis or the intracellular steps of transport. The membrane-active compounds could presumably also change the interaction of the modified G protein with the adenylyl cyclase. Along these lines, it has been demonstrated that the cholesterol content of proteoliposomes is important for the coupling between G^sub s^ and adenylyl cyclase (Bai and Youguo, 1998), and that the cholesterol-binding drug filipin can inhibit forskolin-induced activation of adenylyl cyclase in C6-2B glioma cells (Fagan et al., 2000). Thus, the rate of endocytic uptake should be quantified directly, and not be estimated from the production of cAMP.

The rate of endocytosis of a given toxin can be measured in a number of ways. In the case of ricin, cell-surface bound toxin can easily be removed with lactose, which will compete with the receptor binding. For other toxins such as Shiga toxin and cholera toxin such a method cannot be used, but one can instead use toxins modified for instance with biotin (Torgersen et al., 2001). Earlier studies also employed the addition of antibodies to the ligand (the toxin) to assess whether it is still surface associated. However, it has become clear that antibodies are not able to reach ligands (toxins) in invaginations with narrow necks (Smythe et al., 1992), and such methods might therefore overestimate the amount of toxin internalized.

In many cases the internalization of ligands are studied by measuring the amount taken in during the first 30 minutes. Since some ligands such as transferrin are efficiently recycled whereas other ligands are just accumulating with time, such measurements are not ideal to obtain information as to whether the rate of uptake of a ligand by a certain endocytic mechanism is affected by a compound that has been added. Recycling might also be affected, and one cannot make a direct comparison between ligands with different kinetics and with differences in intracellular sorting. Therefore, measurements of the kinetics of uptake has to be performed. For instance, although a certain compound affects the rate of uptake of transferrin, the total amount of cell-associated transferrin may be unchanged after 30 min incubation if both the rate of uptake and recycling is decreased.

Both endocytosis and intracellular transport can be followed by either electron microscopy or confocal microscopy. Such studies have provided a large amount of information, but there are some pitfalls: The visualization of a ligand in a certain structure at the cell surface, for instance in caveolae, does not mean that the ligand is endocytosed from this structure. It might rather be trapped in that location, for instance in caveolae, which have recently been shown to be stable structures (Thomsen et al., 2002). Furthermore, the toxins are active at so low concentrations that they may not necessarily be seen in an organelle although their transport through this organelle is essential for their action. For instance, ricin is able to enter the ER on its way to the cytosol (Rapak et al., 1997), but has so far not been seen in this location by microscopy.

Entry of toxins into an endosomal compartment

After endocytic uptake the protein toxins can be observed in endosomes. But which endosomal compartments do they enter and by which endocytic mechanisms are they taken up? There are several endocytic mechanisms operating (Conner and Schmid, 2003; Sandvig and van Deurs, 2002), and the toxins seem to exploit the various ports of entry into the cell (Sandvig and van Deurs, 2002). Thus, the primary endocytic vesicles into which the toxins enter are likely to be different and in most cases they are not well characterized. After 5-10 minutes toxins taken in by clathrin-dependent and - independent mechanisms may end up in the same early endosomal compartment labeled with markers such as for instance transferrin or EEA1 (Hansen et al., 1993; Tran et al., 1987). Interestingly, also LacCer, which is internalized by a clathrin-independent but dynamin- dependent pathway becomes colocalize\d with transferrin in human skin fibroblasts (Sharma et al., 2003). However, cholera toxin has in some cells also been found in a separate compartment, the so- called caveosome (acaveolin-1 containing organelle). This is the same compartment that is reached by the virus SV40 after entry from caveolae (Pelkmans et al., 2001). In contrast to SV40, it is not known whether cholera toxin can induce its own uptake from caveolae (Pelkmans et al., 2001; Thomsen et al., 2002). The possibility exists that cholera toxin enters a caveolin-containing intracellular compartment after entry by a caveolae-independent mechanism. Anyhow, cholera toxin can act on cells such as Caco-2 cells that do not have caveolae or caveolin-1, and expression of caveolin-1 and formation of caveolae in these cells do not change the effect of cholera toxin (Torgersen et al., 2001).

The endocytic pathway used by a certain toxin also seems to vary between different cell types. Shiga toxin can be internalized both by clathrin-dependent and -independent pathways, and the fraction of the toxin endocytosed by clathrin-dependent endocytosis seems to depend on the cell line and the conditions under which the cells are grown (our unpublished data and (Sandvig et al., 1989; Schapiro et al., 1998; Nichols, 2002; Lingwood, 1999)). Similarly, the endocytic uptake of cholera toxin can apparently occur by different mechanisms (Orlandi and Fishman, 1998; Shogomori and Futerman, 2001a; Torgersen et al., 2001). The uptake of this toxin has been reported to be raft- dependent, but it can also occur in cells treated with filipin which binds cholesterol (Shogomori and Futerman, 2001a) and in cells treated with methyl-[beta]-cyclodextrin to extract cholesterol (Torgersen et al., 2001). The toxin is endocytosed after methyl- [beta]-cyclodextrin treatment even when clathrin-dependent endocytosis is blocked either by expression of antisense-clathrin or a dominant negative mutant of dynamin which can be expected to block not only uptake from clathrin-coated pits but also from caveolae (Figure 2). Thus, there seem to be raft- and clathrin-independent forms of endocytosis. Also, cholera toxin has been reported to be internalized from clathrin-coated pits in some cells (Torgersen et al., 2001; Shogomori and Futerman, 2001a; Nichols, 2002). However, it should be noted that it is not yet clear whether all the different types of uptake mediate Golgi transport and effect of cholera toxin on the cAMP level in the cells.

Pathways used by toxins between endosomes and the Golgi apparatus

There is now evidence that both ricin and Shiga toxin enter the Golgi apparatus by a Rab9-independent route (Iversen et al., 2001; Sandvig et al., 2002). How they are sorted from early endosomes in the direction of the Golgi is, however, not clear, and the pathways for transport of these two toxins seem to differ. In the case of Shiga B, the binding subunit of Shiga toxin, the t-SNAREs syntaxin 6 and 16 and the v-SNAREs VAMP3 and VAMP4 seem to be important (Mallard et al., 2002). Furthermore, Rab6a' was found to play a role for the step between endosomes and the Golgi apparatus (Mallard et al., 2002). Also, the transport has been reported to be dependent on Rab 11 and clathrin (Johannes and Goud, 2000), and to occur independently of late endosomes (Nichols, 2002; Nichols and Lippincott-Schwartz, 2001; Johannes and Goud, 2000). In agreement with these results Shiga toxin enters the Golgi by a Rab9- independent pathway (Sandvig et al., 2002). In contrast to Shiga toxin, which binds the glycosphingolipid Gb3, after endocytosis LacCer was found to enter the Golgi apparatus by a Rab7 and Rab9- dependent route (Sharma et al., 2003). The reason for these differences is not known. However, the efficiency of Golgi transport can be dependent on the lipid composition (Sandvig et al., 1992; Lingwood, 1999), and the possibility exists that the length of the fatty acids of the glycolipid studied or the lipid composition of the cells studied might affect the result obtained. In the case of ricin, transport to the Golgi apparatus occurs independently of Rab 11 and clathrin (Iversen et al., 2001). Moreover, transport of ricin and Shiga toxin to the Golgi apparatus is differentially regulated by increased cytosolic calcium, which can stimulate ricin transport by a factor of twenty in MDCK II cells whereas the effect on transport of Shiga B is much less (Lauvrak et al., 2002). The presence of subdomains in endosomes has recently been demonstrated (Pfeffer, 2003), and toxins may be localized to different subdomains from where transport is differentially regulated.

Fig. 2. BHK antisense dathrin cells (Iversen et al., 2003) and Hela K44A (Damke et al., 1994) cells were grown with or without tetracycline. Some of the cultures without tetracycline (that is, cultures expressing either antisense clathrin or mutant dynamin) were further incubated for 30 min with methyl-[beta]-cydodextrin (CD) (5 mM) to remove cholesterol. Then TAG-labeled cholera toxin was added, the cells were further incubated for 20 min at 37 C, and the amount of endocytosed toxin was measured as described earlier (Torgersen et al., 2001). Note that cholesterol depletion increases internalization of cholera toxin in the antisense and mutant dynamin- expressing cells (mean S. D.).

After endocytosis of the toxins: What is their likelihood of being transported to the Golgi apparatus? Does it matter whether they are endocytosed by one mechanism or another? It has been demonstrated by several laboratories that different toxins can enter the Golgi apparatus even when they are not internalized from clathrin-coated pits. This was first shown for ricin in 1987 (Sandvig et al., 1987). When clathrin-dependent endocytosis was inhibited by cytosol acidification, ricin internalized under those conditions could enter the Golgi apparatus. Later on it has been found that other conditions, which inhibit uptake from clathrin- coated pits, do not block Golgi transport of cholera toxin, Shiga toxin B and ricin (Torgersen et al., 2001; Nichols and Lippincott- Schwartz, 2001; Iversen et al., 2001). There is evidence that interfering with clathrindependent uptake by expression of clathrin- hubs does not reduce the transport of cholera toxin to the Golgi apparatus, and it was suggested that the clathrin-dependent pathway may not lead to Golgi transport of cholera toxin (Le and Nabi, 2003). On the other hand, it was reported that caveolin-1 overexpression decreased Golgi transport of cholera toxin (Le and Nabi, 2003). Why this is the case is, is not known. Caveolin-1 could somehow interfere directly with endosome-to-Golgi transport. Also, overexpression of caveolin, which is a cholesterol-binding protein, might affect cholesterol transport and localization and thereby change the content of cholesterol in a given organelle. As discussed below this might be important for toxin transport.

A common requirement for Golgi transport of ricin, Shiga toxin and cholera toxin from endosomes is cholesterol. When the level of cholesterol is reduced, also Golgi transport is affected (Grimmer et al., 2000; Shogomori and Futerman, 2001b; Falguieres et al., 2001). Since both Shiga and cholera toxin bind glycolipid receptors, and since also ricin can bind to glycolipids (in addition to glycoproteins), the possibility existed that glycolipid-binding toxins in lipid rafts were preferentially transported to the Golgi apparatus. However, this is not the case. We have recently found that ricin is transported to the Golgi apparatus and intoxicates cells equally well in cells which cannot synthesize glycolipids due to lack of glucosyl-ceramide synthetase as in the cells where the enzyme was backtransfected (Spilsberg et al., 2003). Remarkably, ricin transport to the Golgi is sensitive to cholesterol-depletion also in cells without glycolipids. The cells without glucosylceramide synthetase (GM95) and the cells where the enzyme with an ER (endoplasmic reticulum) retention signal was back- transfected (GM95-CGlcT-KKVK: GM95 backtransfected with Ceramideglucosyl-transferase) were also used to study the importance of glycolipids and glycolipid synthesis for the effect of other toxins. As shown in Figure 3, the protein toxins abrin and modeccin both had the same effect on these cell lines indicating that not only do the toxins not require glycolipids as receptors, but glycolipid synthesis is not required for efficient sorting of the toxins through the endocytic pathway, to the Golgi apparatus and the ER, and finally to the cytosol where the toxic effect is exerted. It is possible that the presence of a toxin-receptor complex in lipid rafts is essential for sorting to the Golgi apparatus. However, it is apparently not important whether the receptor is of protein or lipid nature.

Retrograde transport through the Golgi apparatus and to the ER

A few protein toxins, such as Pseudomonas exotoxin A and cholera toxin, have a KDEL or KDEL-like sequence (in the case of Pseudomonas toxin it has the sequence RDELK which is processed to DELK which binds to the KDEL receptor), and these toxins can therefore be transported retrogradely to the ER after binding to the KDEL receptor, i.e. in COPI-coated vesicles (for review, see (Sandvig and van Deurs, 2002)). However, even cholera toxin B, which is without this sequence, moves retrogradely (Sandvig et al., 1996). Also, several of the other protein toxins that enter the ER before translocation to the cytosol, for instance Shiga toxin and ricin, do not contain the KDEL sequence. A number of investigations support the idea that these toxins can be transported to the ER independently of COPI-coated vesicles. For instance, overexpression of lysozyme-KDEL that saturates the KDEL receptors protects against Pseudomonas toxin, but not against Shiga-like toxin 1 (Jackson et al., 1999). More recent data suggest that Shiga toxin can be transported by a Rab\1-dependent, COPI-independent mechanism from the Golgi to the ER (White et al., 1999; Girod et al., 1999). Interestingly, the transport from Golgi to the ER seems to be dependent on actin (Valderrama et al., 2001; Luna et al., 2002) and movement of vesicles by myosin motors (Duran et al., 2003). When actin is depolymerized by use of latrunculin B, the transport of Shiga toxin B subunit was reported to be inhibited (Valderrama et al., 2001). On the other hand, when actin polymerization is induced by overexpression of Cdc42, also this can inhibit retrograde transport (Luna et al., 2002). Why this is the case is not clear, but it could be due to formation of a dense peri-Golgi actin network that might physically block the movement of vesicles to the ER. Although the retrograde toxin transport is not yet fully understood, studies employing different toxins as tools have demonstrated the usefulness of toxins in the clarification of basic transport mechanisms.

Fig. 3. Effect of abrin and mocleccin on cells with (GM95-CGIcT- KKVK ) and without (GM95) the ability to synthesize glycosphingolipids. The cells were incubated with increasing concentrations of toxins overnight. Then the protein synthesis was measured during a 15 min interval as previously described (Spilsberg et al., 2003) by adding [^sup 3^H]leucine in leucine-free Hepes- medium and measuring the radioactivity incorporated into trichloroacetic acid precipitable material.

Transport of toxins from the ER to the cytosol

After arrival of the protein toxins in the ER the enzymatically active part of the toxins could be released from the rest of the toxin molecule by the help of ER chaperones. For instance, in the case of cholera toxin, the ER protein disulfide isomerase seems to be essential for this process (Tsai et al., 2001). Similar data do not exist for Shiga toxin and ricin, but the scenario might be the same. After release of the enzymatically active part it still has to be transferred to the cytosol to act on the cells. This seems to occur by means of the translocator Sec61 which interacts both with ricin and with cholera toxin (Schmitz et al., 2000; Hazes and Read, 1997; Simpson et al., 1999; Wesche et al., 1999). However, the details of the translocation process are not known, and it is not clear whether also the nonenzymatic part of the toxin, the B- fragments, can be translocated to the cytosol. Even though Shiga B has an effect on cells after expression in the cytosol (Nakagawa et al., 1999) and even though epitopes bound to the B-fragment can be presented at the cell surface by MHC class I (Lee et al., 1998; Haicheur et al., 2000), no direct proof for translocation of the B- fragment through the Sec61 translocator has been presented. Future studies are required to clarify this process.

Conclusions

Clarification of mechanisms involved in uptake and transport of toxins such as Shiga toxin, ricin and cholera toxin is important for a number of reasons. Several of these toxins are still a problem in infectious diseases. Furthermore, the toxins can be exploited as tools in medical therapy. They can be used to selectively eradicate certain cells such as cancer cells, and they can be used to deliver epitopes or other proteins into cells. Importantly, they have proven useful in investigations of basic mechanisms in cell biology.

Acknowledgements. We are grateful to A. G. Myrann for producing the figures in the current article. The work referred to from our own groups was supported by the Norwegian and Danish Cancer Societies, the Norwegian Research Council for Science and Humanities, the Danish Medical Research Council, The Novo Nordisk Foundation, the Jahre Foundation, and Jeanette and Soren Bothners Legacy.

References

Andreoli, S. P., Trachtman, H., Acheson, D. W. K., Siegler, R. L., Obring, T. G.: Hemolytic uremic syndrome: epidemiology, pathophysiology, and therapy. Pediatr. Nephrol. 17, 293-298 (2002).

Bai, L., Youguo, H.: Effect of cholesterol/phospholipid ratio on stimulatory GTP-binding protein function. Biochem. Mol. Biol. Int. 45, 1155-1162 (1998).

Conner, S. D., Schmid, S. L.: Regulated portals of entry into the cell. Nature 422, 37-44 (2003).

Damke, H., Baba, T., Warnock, D. E., Schmid, S. L.: Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127, 915-934 (1994).

Duran, J. M., Valderrama, F., Castel, S., Magdalena, J., Tomas, M., Hosoya, H., Renau-Piqueras, J., Malhotra, V., Egea, G.: Myosin motors and not actin comets are mediators of the actin-based Golgi- to-endoplasmic reticulum protein transport. Mol. Biol. Cell 14, 445- 459 (2003).

Fagan, K. A., Smith, K. E., Cooper, D. M.: Regulation of the Ca^sup 2+^-inhibitable adenylyl cyclase type VI by capacitative Ca^sup 2+^ entry requires localization in cholesterol-rich domains. J. Biol. Chem. 275, 26530-26537 (2000).

Falguieres, T., Mallard, F., Baron, C., Hanau, D., Lingwood, C., Goud, B., Salamero, J., Johannes, L.: Targeting of shiga toxin b- subunit to retrograde transport route in association with detergent- resistant membranes. Mol. Biol. Cell 12, 2453-2468 (2001).

Frankel, A. E., Kreitman, R. J., Sausville, E. A.: Targeted toxins. Clin. Cancer Res. 6, 326-334 (2000).

Girod, A., Storrie, B., Simpson, J. C., Johannes, L., Goud, B., Roberts, E. M., Lord, J. M., Nilsson, T., Pepperkok, R.: Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum. Nat. Cell Biol. 1, 423-430 (1999).

Grimmer, S., Iversen, T. G., van Deurs, B., Sandvig, K.: Endosome to Golgi transport of ricin is regulated by cholesterol. Mol. Biol. Cell 11, 4205-4216 (2000).

Gura, T.: Magic bullets hit the target. Nature. 417, 584-586 (2002).

Haicheur, N., Bismuth, E., Bosset, S., Adotevi, O., Warmer, G., Lacabanne, V., Regnault, A., Desaymard, C., Amigorena, S., Ricciardi- Castagnoli, P., Goud, B., Fridman, W. H., Johannes, L., Tartour, E.: The B subunit of Shiga toxin fused to a tumor antigen elicits CTE and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens. J. Immunol. 165, 3301-3308 (2000).

Hansen, S. H., Sandvig, K., van Deurs, B.: Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors. J. Cell Biol. 123, 89-97 (1993).

Hazes, B., Read, R. J.: Accumulating evidence suggests that several AB-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells. Biochemistry 36, 11051-11054 (1997).

Iversen, T.-G., Skretting, G., Elorente, A., Nicoziani, P., van Deurs, B., Sandvig, K.: Endosome to Golgi transport of ricin is independent of clathrin and of the Rab9- and Rab11-GTPases. Mol. Biol. Cell 12, 2099-2107 (2001).

Iversen, T.-G., Skretting, G., van Deurs, B., Sandvig, K.: Formation of clathrin-coated pits with long dynamin-wrapped necks upon inducible expression of antisense to clathrin. Proc. Natl. Acad. Sci. USA 100, 5175-5180 (2003).

Jackson, M. E., Simpson, J. C., Girod, A., Pepperkok, R., Roberts, L. M., Lord, J. M.: The KDEL retrieval system is exploited by Pseudomonas exotoxin A, but not by Shiga-like toxin-1, during retrograde transport from the Golgi complex to the endoplasmic reticulum. J. Cell Sci. 112, 467-475 (1999).

Johannes, L., Goud, B.: Facing inward from compartment shores: How many pathways were we looking for? Traffic 1, 119-123 (2000).

Kreitman, R. J.: Toxin-labeled monoclonal antibodies. Curr. Pharm. Biotechnol. 2, 313-325 (2001).

Kreitman, R. J.: Recombinant toxins for the treatment of cancer. Curr. Opin. Mol. Ther. 5, 44-51 (2003).

Lauvrak, S. U., Llorente, A., Iversen, T.-G., Sandvig, K.: Selective regulation of the Rab9-independent transport of ricin to the Golgi apparatus by calcium. J. Cell Sci. 115, 3449-3456 (2002).

Le, P. U., Nabi, I. R.: Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum. J. Cell Sci. 116, 1059-1071 (2003).

Lee, R.-S., Tartour, E., van der Bruggen, P., Vantomme, V., Joyeux, I., Goud, B., Fridman, W. H., Johannes, L.: Major histocompatibility complex class I presentation of exogenous tumor antigen fused to the Bfragment of Shiga toxin. Eur. J. Immunol. 28, 2726-2737 (1998).

Lingwood, C. A.: Glycolipid receptors for verotoxin and Helicobacter pylori: role in pathology. Biochim. Biophys. Acta 1455, 375-386 (1999).

Luna, A., Matas, O. B., Martinez-Menarguez, J. A., Mato, E., Duran, J. M., Ballesta, J., Way, M., Egea, G.: Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP. Mol. Biol. Cell 13, 866-879 (2002).

Mallard, F., Tang, B. L., Galli, T., Tenza, D., Saint-Pol, A., Yue, X., Antony, C., Hong, W., Goud, B., Johannes, L.: Early/ recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform. J. Cell Biol. 156, 653-664 (2002).

Nakagawa, I., Nakata, M., Kawabata, S., Hamada, S.: Regulated expression of Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells. Mol. Microbiol. 33, 1190-1199 (1999).

Nichols, B. J.: A distinct class of endosome mediates clathrin- independent endocytosis to the Golgi complex. Nat. Cell Biol. 4, 374- 378 (2002).

Nichols, B. J., Lippincott-Schwartz, J.: Endocytosis without clathrin coats. Trends Cell Biol. 11, 406-412 (2001).

Noakes, K. L., Teisserenc, H. T., Lord, J. M., Dunbar, P. R., Cerundolo, V., Roberts, L. M.: Exploiting retrograde transport of Shiga-like toxin 1 for the delivery of exogenous antigens into the MHC class I presentation pathway. FEBS Lett. 453, 95-99 (1999).

Orlandi, P. A., Fishman, P. H.: Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J. Cell Biol. 141, 905-915 (1998).

Paton, J. C., Paton, A. W.: Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coll infections. Clin. Microbiol. Rev. 11, 450-479 (1998).

Pelkmans, L., Kartenb\eck, J., Helenius, A.: Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat. Cell Biol. 3, 473-483 (2001).

Pfeffer, S.: Membrane domains in the secretory and endocytic pathways. Cell 112, 507-517 (2003).

Rapak, A., Falnes, P. O., Olsnes, S.: Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol. Proc. Natl. Acad. Sci. USA 94, 3783-3788 (1997).

Sandvig, K., Garred, O., Prydz, K., Kozlov, J. V., Hansen, S. H., van Deurs, B.: Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum. Nature 358, 510-511 (1992).

Sandvig, K., Garred, O., van Deurs, B.: Regulated transport of cholera toxin to the endoplasmic reticulum: Correlation with cAMP production. Proc. Natl. Acad. Sci. USA 93, 12339-12343 (1996).

Sandvig, K., Grimmer, S., Lauvrak, S. U., Torgersen, M. L., Skretting, G., van Deurs, B., Iversen, T.-G.: Pathways followed by ricin and Shiga toxin into cells. Histochem. Cell Biol. 117, 131- 141 (2002).

Sandvig, K., Olsnes, S., Brown, J. E., Petersen, O. W., van Deurs, B.: Endocytosis from coated pits of Shiga toxin: a glycolipid- binding protein from Shigella dysenteriae 1. J. Cell Biol. 108, 1331- 1343 (1989).

Sandvig, K., Olsnes, S., Petersen, O. W., van Deurs, B.: Acidification of the cytosol inhibits endocytosis from coated pits. J. Cell Biol. 105, 679-689 (1987).

Sandvig, K., van Deurs, B.: Membrane traffic exploited by protein toxins. Annu. Rev. Cell Dev. Biol. 18, 1-14 (2002).

Schapiro, F. B., Lingwood, C., Furuya, W., Grinstein, S.: pH- independent retrograde targeting of glycolipids to the Golgi complex. Am. J. Physiol. 274, C319-C332 (1998).

Schiavo, G., van der Goot, F. G.: The bacterial toxin toolkit. Nat. Rev. Mol. Cell Biol. 2, 530-537 (2001).

Schmitz, A., Herrgen, H., Winkeler, A., Herzog, V.: Cholera toxin is exported from microsomes by the Sec61p complex. J. Cell Biol. 148, 1203-1212 (2000).

Sharma, D. K., Choudhury, A., Singh, R. D., Wheatley, C. L., Marks, D. L., Pagano, R. E.: Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling. J. Biol. Chem. 278, 7564-7572 (2003).

Shogomori, H., Futerman, A. H.: Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism. J. Biol. Chem. 276, 9182-9188 (2001a).

Shogomori, H., Futerman, A. H.: Cholesterol depletion by methyl- beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons. J. Neurochem. 78, 991- 999 (2001b).

Simpson, J. C., Roberts, L. M., Romisch, K., Davey, J., Wolf, D. H., Lord, J. M.: Ricin A chain utilises the endoplasmic reticulum- associated protein degradation pathway to enter the cytosol of yeast. FEBS Lett. 459, 80-84 (1999).

Smythe, E., Redelmeier, T. E., Schmid, S. L.: Receptor-mediated endocytosis in semiintact cells. Methods Enzymol. 219, 223-234 (1992).

Spilsberg, B., van Meer, G., Sandvig, K.: Role of lipids in the retrograde pathway of ricin intoxication. Traffic 4, 544-552 (2003).

Thomsen, P., Roepstorff, K., Stahlhut, M., van Deurs, B.: Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell 13, 238-250 (2002).

Torgersen, M. L., Skretting, G., van Deurs, B., Sandvig, K.: Internalization of cholera toxin by different endocytic mechanisms. J. Cell Sci. 114, 3737-3747 (2001).

Tran, D., Carpentier, J. L., Sawano, F., Gorden, P., Orci, L.: Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway. Proc. Natl. Acad. Sci. USA 84, 7957-7961 (1987).

Tsai, B., Rodighiero, C., Lencer, W. I., Rapoport, T. A.: Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell 104, 937-948 (2001).

Valderrama, F., Duran, J. M., Babia, T., Barth, H., Renau- Piqueras, J., Egea, G.: Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells. Traffic 2, 717-726 (2001).

Wesche, J., Rapak, A., Olsnes, S.: Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol. J. Biol. Chem. 274, 3443-3449 (1999).

White, J., Johannes, L., Mallard, F., Girod, A., Grill, S., Reinsch, S., Keller, P., Tzschaschel, B., Echard, A., Goud, B., Stelzer, H. K.: Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells. J. Cell Biol. 147, 743-760 (1999).

Kirsten Sandvig(a), Bjorn Spilsberg(a), Silje U. Lauvrak(a), Maria L. Torgersen(a), Tore-Geir Iversen(a), Bo van Deurs(b)

a Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway

b Structural Cell Biology Unit, Department of Anatomy, The Panum Institute; University of Copenhagen, Copenhagen, Denmark

Corresponding author: Kirsten Sandvig, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. Phone: +4722934294, Fax: +4722508692, E-mail: ksandvig@radium.uio.no

Copyright Urban & Fischer Verlag Apr 2004

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