Retrograde Transport of Pseudomonas Exotoxin a by the KDEL Receptor 469 Prepared Locally

Journal of Cell Science 112, 467-475 (1999) 467 Printed in Great Britain © The Company of Biologists Limited 1999 JCS4636 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 Michelle E. Jackson1,*, Jeremy C. Simpson1,*, Andreas Girod2,*, Rainer Pepperkok2, Lynne M. Roberts1 and J. Michael Lord1,‡ 1Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK 2Light Microscopy Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK *All three authors contributed equally to this study ‡Author for correspondence (e-mail: [email protected]) Accepted 27 November 1998; published on WWW 25 January 1999 SUMMARY To investigate the role of the KDEL receptor in the retrieval when they express additional KDEL receptors. These data of protein toxins to the mammalian cell endoplasmic suggest that, in contrast to SLT-1, PE can exploit the KDEL reticulum (ER), lysozyme variants containing AARL or receptor in order to reach the ER lumen where it is believed KDEL C-terminal tags, or the human KDEL receptor, have that membrane transfer to the cytosol occurs. This been expressed in toxin-treated COS 7 and HeLa cells. contention was confirmed by microinjecting into Vero cells Expression of the lysozyme variants and the KDEL antibodies raised against the cytoplasmically exposed tail receptor was confirmed by immunofluorescence. When of the KDEL receptor. Immunofluorescence confirmed that such cells were challenged with diphtheria toxin (DT) or these antibodies prevented the retrograde transport of the Escherichia coli Shiga-like toxin 1 (SLT-1), there was no KDEL receptor from the Golgi complex to the ER, and this observable difference in their sensitivities as compared to in turn reduced the cytotoxicity of PE, but not that of SLT- cells which did not express these exogenous proteins. By 1, to these cells. contrast, the cytotoxicity of Pseudomonas exotoxin A (PE) is reduced by expressing lysozyme-KDEL, which causes a redistribution of the KDEL receptor from the Golgi Key words: Retrograde transport, KDEL receptor, Pseudomonas complex to the ER, and cells are sensitised to this toxin exotoxin A, Endoplasmic reticulum INTRODUCTION (see reviews by Sandvig and van Deurs, 1994; Lord et al., 1994; Lord and Roberts, 1998). The ER is an attractive Toxins such as ricin, Pseudomonas exotoxin A (PE), diphtheria contender for a translocation compartment as it contains factors toxin (DT), Shiga toxin (ST) and Escherichia coli Shiga-like which could facilitate entry to the cytosol by reducing and toxin 1 (SLT-1), kill cells by inhibiting protein synthesis. The unfolding the toxins prior to their membrane partitioning. It intoxication process can be divided into four main stages: cell also contains protein transport machinery, usually thought to binding, endocytosis, membrane translocation to the cytosol be involved in the translocation of newly synthesised proteins and, finally, enzymatic modification of a cytosolic target. DT into the ER lumen, but which, as in the case of the Sec61p and PE inhibit protein synthesis by catalysing the ADP- complex, has also been implicated in reverse translocation of ribosylation of elongation factor 2 (EF-2) (Pappenheimer, misfolded proteins from the ER lumen into the cytosol (Wiertz 1977; Iglewski and Kabat, 1975), whereas ricin, ST and SLT- et al., 1996; Pilon et al., 1997; Plemper et al., 1997). 1 hydrolytically cleave a specific N-glycosidic bond in the A number of studies have provided evidence to support the large rRNA of the 60S subunit of 80S ribosomes (Endo et al., physiological routing of certain toxins to the ER of mammalian 1987). The consequent release of a single adenine residue from cells. Firstly, brefeldin A (BFA) treatment, which disrupts the the highly conserved target rRNA sequence renders the Golgi stack causing it to merge with the ER, protects cells ribosome incapable of protein synthesis. In order to reach their against PE, ricin and SLT-1, but not against DT (Yoshida et al., cytosolic targets, the toxins, once internalised by the cell, must 1991; Sandvig et al., 1991a). BFA does not block endocytosis cross an intracellular membrane. Unlike DT, which enters the nor does it prevent the toxins from reaching the trans-Golgi cytosol from acidified endosomes, the intracellular site of network (TGN) in the cell lines tested (Yoshida et al., 1991; translocation of PE, ricin and ST/SLT-1 is thought to be the ER Sandvig et al., 1991a). Epidermal cell differentiation factor, an 468 M. E. Jackson and others ADP-ribosyltransferase from Staphylococcus aureus E-1, for the cytotoxicity of PE (Chaudhary et al., 1990), and its causes a similar breakdown of the Golgi to that observed with replacement with KDEL (Seetharam et al., 1991) even BFA, and this metabolite also protects cells from the effects of enhances its potency. These data suggest that PE may directly ricin (Sugai et al., 1992a,b). Secondly, HRP conjugates of ST interact with the KDEL receptor, a view supported by the in have been directly visualised in the ER lumen of cells that have vitro association with immobilised KDEL receptors (Kreitman been sensitised to the toxin (Sandvig et al., 1992; 1994). and Pastan, 1995), although this has never been convincingly Indeed, the ST B fragment alone can be transported to the ER demonstrated in vivo. Endocytosed CT has also been visualised (Kline and Lingwood, 1994; Sandvig et al., 1994). Thirdly, in the Golgi and the ER (Sandvig et al., 1996), and an overexpression of trans-dominant mutant GTPases involved in immunofluorescence study demonstrated the retrograde regulating vesicle traffic between the ER and the Golgi transport of endocytosed CT from the Golgi to the ER (Majoul complex significantly reduces the cytotoxic potency of PE, et al., 1996). While it seems likely that CT, with a KDEL ricin and SLT-1, suggesting the importance of transport to or tetrapeptide, is also transported from the Golgi to the ER by through the Golgi stack (Simpson et al., 1995). Finally, interacting with the KDEL receptor, once again this has not modified forms of ST and ricin have been shown, by been directly demonstrated. acquisition of core oligosaccharides, to enter the ER after Involvement of the KDEL retrieval system with the transport endocytosis from the cell surface (Johannes et al., 1997; Rapak of other toxins is, however, less clear. For example, cholera B et al., 1997). chain (which lacks a KDEL sequence; the KDEL of CT is on Should PE, ricin and ST/SLT-1 reach the cytosol from the the A chain) has been visualised in all Golgi cisternae (Sandvig ER, the question remains as to the mechanism of retrograde et al., 1994) and ST, which also lacks a KDEL-like motif, has transport. Following uptake by clathrin-dependent or clathrin- been seen in the Golgi and the ER of sensitised A431 cells independent endocytosis (Sandvig et al., 1991b; Simpson et al., (Sandvig et al., 1992, 1994). Furthermore, removal of the 1998), the toxins are delivered to endosomes, and a large RDEL sequence from LT was reported to have no effect on its proportion is subsequently delivered to lysosomes for potency (Ciepak et al., 1995), although a different study degradation (reviewed in Sandvig and van Deurs, 1996). suggested that disruption of the LT-RDEL did indeed slow However, a small fraction of internalised ricin and ST has been down the toxin-induced secretion of chloride ions (Lencer et visualised in the TGN (van Deurs et al., 1988). Such toxin is al., 1995). Like ST and SLT-1, ricin does not contain a KDEL most likely in association with the membrane having exploited or KDEL-like motif. However, the fact that addition of a KDEL a route utilised by, for example, unoccupied mannose-6- tag to ricin A-chain increases its cytotoxic effect (Wales et al., phosphate receptors, TGN38 or furin (Goda and Pfeffer, 1988; 1993) at least suggests that a fraction of internalised ricin must Bos et al., 1993; Schafer et al., 1995). That the TGN represents encounter and, in this case, interact with recycling KDEL- a port for subsequent transport steps is indicated by the receptors, and that such interaction facilitates eventual delivery correlation between an intact Golgi and sensitivity to ricin and to the cytosol. ST (Sandvig et al., 1991a; Yoshida et al., 1991), and the In the present study we have investigated in vivo whether protective effects of low temperatures known to block the the KDEL retrieval system plays a part in the intracellular delivery of toxins to this compartment (van Deurs et al., 1987; transport of SLT-1 and PE. Cells were treated in ways that Sandvig et al., 1989). affected the cellular level, intracellular distribution, or In addressing retrograde transport from the TGN to the ER, functioning of the KDEL receptor. The predicted modifications it has been postulated that the toxins may exploit an ER were shown to have occurred by immunofluorescence. The retrieval system (Pelham et al., 1992). There are a number of effects of these modifications on the susceptibility of the cells signals which function as retrieval markers for escaped resident to intoxication demonstrate that PE exploits the KDEL retrieval ER proteins in the Golgi compartments. Type I and type II system to reach the ER, whereas SLT-1 utilises a different transmembrane resident ER proteins contain cytosolically carrier to reach the same destination. located sequences at their C and N termini, respectively, which act as ER retrieval signals (Jackson et al., 1993; Schutze et al., 1994). The only retrieval signal that has been identified for MATERIALS AND METHODS resident lumenal proteins is the K/RDEL (HDEL in yeast) motif found at the C terminus of soluble ER proteins (Munro Materials and Pelham, 1987).

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