©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ricin Cytotoxicity Is Sensitive to Recycling between the Endoplasmic Reticulum and the Golgi Complex (*)

(Received for publication, May 12, 1995 )

Jeremy C. Simpson (1)(§) Christiane Dascher(§) (2) Lynne M. Roberts (1) J. Michael Lord (1) William E. Balch (2)

From the  (1)Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom and the (2)Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cytotoxic proteins that kill mammalian cells by catalytically inhibiting protein synthesis must enter the cytosol in order to reach their substrates. With the exception of diphtheria toxin, which enters the cytosol from acidified endosomes, the intracellular site of translocation of other toxins including ricin, Escherichia coli Shiga-like toxin-1, and Pseudomonas exotoxin A is likely to involve early compartments of the secretory pathway. We have used a molecular approach to identify the site and mechanism of toxin delivery to the cytosol by transiently expressing mutant GTPases that inhibit the assembly of biochemical complexes mediating anterograde and retrograde transport in the exocytic and endocytic pathways. The results provide evidence to suggest that receptors actively recycling between the endoplasmic reticulum and terminal Golgi compartments are essential for toxin translocation to the cytosol from the endoplasmic reticulum. The rapid kinetics of intoxication demonstrate a substantial level of bidirectional membrane flow and sorting through the early secretory pathway.


INTRODUCTION

Transport of proteins from the cell surface to endocytic compartments and the trans-Golgi network (TGN) (^1)involves receptors that actively recycle via clathrin-dependent or clathrin-independent mechanisms(1) . In contrast, the membrane dynamics mediating retrograde transport from the TGN to the ER remain to be clarified. Insight into this problem has been limited by the availability of marker proteins that efficiently traverse this pathway. Cytotoxic proteins including diphtheria toxin, Escherichia coli Shiga-like toxin-1 (SLT-1), and Pseudomonas exotoxin A, which kill mammalian cells by catalytically inhibiting protein synthesis, enter the cytosol from intracellular compartments in order to reach their substrates. With the exception of diphtheria toxin, which enters the cytosol from acidified endosomes, the intracellular site of translocation of the other toxins is now believed to involve either Golgi compartments or the endoplasmic reticulum (ER) (reviewed in (2) and (3) ).

Given that the biochemical mechanisms involved in retrograde transport from the cell surface to early compartments of the exocytic secretory pathway are unknown, we have used a molecular approach to address the mechanism and site of toxin release. Vesicle transport of cargo from the ER to Golgi compartments is now recognized to involve two protein complexes, referred to as COPI and COPII, which mediate vesicle budding from either the ER (COPII), or pre-Golgi intermediates and Golgi compartments (COPI) (reviewed in (4) ). The assembly of COPII and COPI coats are regulated by the small GTPases Sar1 and ARF1, respectively (reviewed in (5) ). Vesicle fusion appears to be controlled by a common mechanism involving SNARE complexes (reviewed in (4) ) whose assembly is controlled by GTPases belonging to the Rab family (reviewed in (5) ). By transiently expressing mutant forms of these GTPases in the presence of toxin, we now establish a molecular requirement for COPI, COPII, and SNARE complexes in mediating cytotoxicity. These results lead us to suggest that the retrograde transport of toxins occurs via the active recycling of toxin-specific receptors between the ER and the terminal compartments of the Golgi stack.


MATERIALS AND METHODS

Toxins

-Ricin A chain was purchased from Zeneca Pharmaceuticals (Alderley Edge, United Kingdom). Ricin A chain was (10 µg) was mixed with 10 µg of ricin B chain (Inland Laboratories, Austin, TX) in a total volume of 200 µl in PBS by gently agitating on a klinostat at room temperature for 3 h. A 0.5-ml polyacrylamide column containing immobilized galactose (Pierce) was used to purify reassociated ricin holotoxin from free A chain. The purity of preparations was quantified by SDS-polyacrylamide gel electrophoresis and densitometry. Pseudomonas exotoxin A was obtained from Sigma. Diphtheria toxin was obtained from Calbiochem (La Jolla, CA). SLT-1 was a kind gift from Nicholas Lea, University of Warwick. Briefly, the SLT operon was amplified by PCR from E. coli 0157 cell paste and cloned into a pUC19 expression vector. E. coli JM105 was transformed with this vector and used to express SLT-1 to a high level following isopropyl-1-thio-beta-D-galactopyranoside induction. The cells were washed, and the periplasm fraction extracted and then loaded on a globotriose-Sepharose affinity column (kindly provided by D. Muller, University of Warwick). Pure fractions of SLT-1 were eluted under denaturing conditions of 6 M guanidine-HCl, pH 6.7. These were dialyzed against PBS, and toxin was quantified.

Transient Expression

Transient expression in HeLa cells was carried out as described(6) . Briefly, HeLa cells (7 10^6 cells) were plated in 100-mm dishes the day prior to transfection. After infection with vaccinia T7 polymerase recombinant virus (vTF7-3), the cells were transfected with 7.5 µg of an expression vector carrying VSV-G under control of the T7 promoter (pAR-G) and 50 µl of the transfection reagent LipofectAMINE (Life Technologies, Inc.) in Dulbecco's minimal essential medium (DMEM). After 2 h of transfection at 37 °C, the transfection medium was removed. The cells were detached from the dish with phosphate-buffered saline (PBS) containing 5 mM EDTA, washed once with DMEM, and resuspended in the transfection medium. Fetal bovine serum was added to a final concentration of 2.5%. Cells were plated in 35-mm dishes (1.0 10^6 cells/dish) and incubation continued for 3 h. Cells were washed once with PBS, and ricin holotoxin was added in DMEM, 2.5% serum to individual dishes at concentrations indicated (in duplicate). Cells were then incubated in the presence of toxin for 3 h at 37 °C. Following intoxication, the level of VSV-G synthesis was quantitated by a pulse-chase experiment. Cells were incubated for 15 min in methionine-deficient minimal essential medium, radiolabeled for 10 min with 33 µCi of TranS-label (ICN Biomedicals, Inc., Irvine, CA), and then chased for 5 min in DMEM containing 10% serum. Cells were lysed and VSV-G immunoprecipitated as described(6) . Immunoprecipitations were subsequently analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography(6) . Autoradiograms were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The levels of VSV-G synthesis in toxin-treated cells is reported as percentage of total found in control cells that were not treated with toxin.

Analysis of Transport of VSV-G to the Cell Surface

The rate of VSV-G transport from the ER to the cell surface was determined as follows; HeLa cells (6 10^6 cells) were plated in 100-mm dishes the day prior to transfection. After infection of the cells with recombinant vaccinia virus (vTF7-3)(6) , cells were transfected with an expression plasmid encoding the temperature-sensitive mutant form of VSV-G (tsO45) for 4 h at the restrictive temperature (39.5 °C) to retain VSV-G tsO45 in the ER (7, 8) as described(6) . Cells were then rapidly transferred to a 40 °C water bath, washed twice with PBS prewarmed to 40 °C, and detached with PBS containing 5 mM EDTA at 40 °C. Cells were washed once with ice-cold PBS containing 1 mM MgCl(2) and 1 mM CaCl(2) and resuspended in ice-cold minimal essential medium, 2.5% serum to a cell density of 1 10^6 cells/ml. For each time point, 1 ml of the cell suspension was transferred into a 5-ml Falcon tube and cells were incubated at the permissive temperature (32 °C) for the indicated time to allow the synchronized release of tsO45 VSV-G from the ER and transport to the cell surface. At the indicated time points under ``Results and Discussion,'' cells were transferred to ice and diluted in ice-cold PBS containing 0.5% bovine serum albumin and 0.02% NaN(3). Transport of VSV-G to the cell surface was subsequently determined by fluorescence-activated cell sorting analysis using an antibody specific for the extracytoplasmic domain of VSV-G (BW8G5) and visualized by a fluorescein isothiocyanate-conjugated anti-mouse antibody.


RESULTS AND DISCUSSION

To define the molecular basis for retrograde transport from the TGN to the ER in vivo, we applied a transient expression system to overproduce trans dominant interfering mutants of GTPases involved in vesicular traffic in both the exocytic and endocytic pathways to assess their effect on the toxicity of ricin and related toxic proteins. Toxin action was measured by co-expressing each GTPase with a plasmid encoding vesicular stomatitis virus glycoprotein (VSV-G), an N-glycosylated type 1 membrane protein. The ability of increasing concentrations of ricin and other toxins to inhibit the synthesis of VSV-G was examined after 5-6 h of transient expression where GTPases are expressed 5-15-fold above the endogenous level(6, 9) . The effects of increasing concentrations of ricin on the synthesis of VSV-G in the absence of GTPases is shown in Fig. 1. Half-maximal toxicity was observed at a concentration of 2 ng/ml, with greater than 90% inhibition at concentrations above 25 ng/ml. These results are similar to the effect of ricin on general protein synthesis in vivo and in vitro as reported previously(10, 11) .


Figure 1: Effect of ricin on VSV-G synthesis in cells transiently expressing protein in the absence of mutant GTPases. Transient expression of VSV-G in HeLa cells and exposure to ricin was carried out as described under ``Materials and Methods.''



We first examined the effects of a dynamin element 1 mutant that strongly inhibits clathrin-mediated endocytosis(12) , a Rab5 mutant that inhibits receptor recycling from endosomes to the cell surface (13) , and a Rab9 mutant that inhibits clathrin-mediated transport of the mannose-6-phosphate receptor from the late endosome to the TGN (14) . None of these mutants prevented either ricin or diphtheria toxicity (Table 1). While indirect experiments have previously suggested that diphtheria toxin gains access to endosomes via a clathrin-mediated pathway(15) , these results demonstrate that both classes of toxins can be efficiently internalized via receptor recycling pathways that are clathrin-independent.



Since modified forms of ricin, which cannot bind galactose-bearing receptors but which contain high mannose oligosaccharides enabling them to be internalized via the mannose receptor to the TGN, fail to intoxicate macrophages(16) , ricin intoxication of these cells apparently requires binding to an intracellular galactosylated component which itself is capable of undergoing retrograde transport through the Golgi stack. By analogy to endocytic receptors, such a receptor(s) may be actively recirculating between one or more compartments of the early secretory pathway. A test for this possibility is to determine if the toxicity of ricin can be prevented by inhibiting the anterograde transport of the putative receptor(s) to its site of interaction with ricin in the TGN. In principle, such a block would deplete recycling receptor(s) from terminal Golgi compartments, therefore reducing toxin sensitivity. For this purpose, we examined the ability of dominant interfering mutants of Rab1, ARF1, and Sar1, small GTPases that regulate the assembly of protein complexes involved in vesicle formation, targeting, and fusion in the early exocytic pathway to prevent ricin cytotoxicity (reviewed in (5) ). Mutant forms of these GTPases defective in guanine nucleotide exchange and/or hydrolysis have potent inhibitory effects on the anterograde transport of VSV-G through the secretory pathway(6, 9, 17, 18, 19, 20, 21, 22) . For these experiments, VSV-G was used not only as a translational marker as shown above (Fig. 1) but, in addition, as a measure for inhibition of anterograde transport between the ER and the Golgi stack. This was accomplished by following the processing of VSV-G N-linked oligosaccharides to the endoglycosidase H-resistant forms, a hallmark of delivery to the cis/medial Golgi compartments (21, 22) .

Rab1 has been proposed to be involved in a late vesicle docking/fusion step (18) requiring the formation of SNARE complexes(23, 24) . Overexpression of Rab1a mutants that inhibit the targeting/fusion of ER- and Golgi-derived vesicular carriers (9, 18, 19) markedly protected cells from ricin intoxication (Fig. 2A). The Rab1a(N124I)-activated mutant, which has a high rate of guanine nucleotide exchange(18) , allowed VSV-G synthesis at 62% of the control value observed in the presence of 5 ng/ml ricin (Fig. 2A), a concentration that allowed only 22% synthesis in the absence of the GTPase mutant (Fig. 1). This degree of protection correlates well with the ability of the mutant to inhibit VSV-G transport by 64% (Fig. 3c). The Rab1a(S25N) mutant restricted to the GDP-bound form (19) was less effective, 5 ng/ml ricin allowing VSV-G synthesis at 39% of the control (Fig. 2A) and causing a corresponding 39% inhibition of VSV-G transport (Fig. 3d). In contrast, overexpression of wild type Rab1a, which has no effect on VSV-G transport (Fig. 3b), did not protect against ricin, allowing VSV-G synthesis at only 12% of the level observed in the absence of ricin (Fig. 2A). At the highest ricin concentration tested (100 ng/ml), a level that completely inhibits cellular protein synthesis in the control (Fig. 1), both the S25N and N124I mutants still allowed VSV-G synthesis to 33% of the level observed in the absence of toxin (Fig. 2A). These values are summarized in Table 1. This level of protection is striking given that only one molecule of ricin is necessary to inhibit cellular protein synthesis(3) , suggesting that a significant fraction of the cells have completely inhibited the transport of ricin to its site of release. Identical results were observed with E. coli SLT-1 and Pseudomonas exotoxin A, while diphtheria toxin was completely insensitive to overexpression of both Rab1 mutants at all concentrations tested (data not shown). These data provide evidence that the assembly of fusion complexes known to be involved in anterograde traffic is required for the retrograde transport of ricin from the TGN to its site of release in an early compartment of the secretory pathway, emphasizing the possible need for recycling receptors.


Figure 2: Ricin toxicity is inhibited by overexpression of trans dominant negative mutants of Rab1a, ARF1, and Sar1. Shown are the autoradiograms (upperpanels) and quantitation (lowerpanels) of the effects of ricin intoxication on VSV-G synthesis in cells co-expressing the indicated wild-type (wt) and mutant forms of Rab1a (A), ARF1 (B), and Sar1 (C). Transient expression and toxin treatment were performed as described under ``Materials and Methods'' and co-transfection of VSV-G with the indicated GTPase as described earlier(6) .




Figure 3: Levels of inhibition of VSV-G transport by trans dominant GTPase mutants. Transfected cells used in the experiment described in Fig. 2were analyzed for the level of processing of VSV-G to the endoglycosidase H-resistant forms as an index of ER to Golgi transport as described(6) .



We next examined the requirement for non-clathrin COPI-coated vesicles in ricin-mediated inhibition of protein synthesis. While a large body of evidence has accrued to suggest a role for COPI coats in anterograde transport through the early secretory pathway (reviewed in (25) ), recent studies have suggested that COPI components may, in addition, mediate retrograde traffic from the cis-Golgi compartment to the ER (26). The assembly of COPI vesicle coats is controlled by ARF1 (reviewed in (27) ). For example, overexpression of GDP- and GTP-restricted mutants of ARF1 potently inhibit VSV-G transport in vivo(6) . A specific prediction is that ARF1 mutants should effectively block ricin intoxication by blocking either anterograde or retrograde transport. Overexpression of the ARF1 mutants restricted to either the GTP (ARF1(Q71L))- or GDP (ARF1(T31N))-bound forms led to marked protection against ricin (Fig. 2B) and inhibition of VSV-G transport (Fig. 3, f and g). No effect was observed with wild-type ARF1 with VSV-G synthesis allowed at only 10% of the value observed in the absence of ricin (Fig. 2B). In particular, in the presence of the ARF1(T31N-``GDP'') mutant VSV-G synthesis was allowed at a level of 50% in the presence of 5 ng/ml toxin (Fig. 2B). The effects of ARF mutants at medium (5 ng/ml) and high (100 ng/ml) doses are summarized in Table 1. Given that brefeldin A (BFA) also inhibits ricin intoxication(28) , and that both BFA and the ARF1(T31N-``GDP'') mutant trigger the loss of COPI coats and the fusion of early cis/medial Golgi compartments with the ER(6, 29) , these data provide strong evidence that components of the COPI coat machinery are essential for the normal routing of a ricin receptor(s) through the Golgi to its site of translocation to the cytosol.

Must ricin be transported from the cis-Golgi compartment to the ER for toxicity? Since both Rab1 and ARF1 mutants inhibit ER to Golgi as well as intra-Golgi transport (6, 22) and therefore cannot address this issue, we examined the effects of dominant interfering mutants of Sar1. Sar1 is essential for the formation of COPII vesicle coats involved in export from the ER(30) . Sar1 has been localized to transitional elements of the ER and is exceptionally abundant on ER-derived, but not Golgi-derived vesicular carriers(20) . Mutants in the COPII machinery that effectively inhibit export from the ER (31) do not inhibit retrograde traffic between the cis-Golgi compartment and the ER in yeast(26) . However, overexpression of a Sar1 GTP-restricted mutant potently inhibits transport from the ER to the Golgi stack in yeast(32) . In mammalian cells, both the GDP-restricted Sar1(T39N) mutant and the GTP-restricted Sar1(H79G) inhibit ER to Golgi transport, but not the retrograde recycling of marker proteins from the Golgi back to the ER(20) . (^2)

As shown in Fig. 2C, overexpression of the Sar1-GTP and Sar1-GDP mutants resulted in a pronounced 54% and 43% protection, respectively, from ricin toxicity at 5 ng/ml, values consistent with their ability to inhibit VSV-G transport by 60% and 33%, respectively (Fig. 3, i and j; Table 1). Similar results were observed for the effects of the Sar1-GTP mutant on intoxication by both E. coli SLT-1 and Pseudomonas exotoxin A (Table 1). The striking sensitivity of SLT-1 to Sar1 mutants suggests that recycling glycosphingolipid receptors, which bind SLT-1(33) , may also gain access to the ER via retrograde transport through the Golgi stack. Importantly, the Sar1-GTP mutant had no significant effect on intoxication of cells with diphtheria toxin (Table 1). Given the specificity of Sar1 function for anterograde transport from the ER, these results suggest that translocation of these potent toxins to the cytosol involves a receptor actively recycling between the ER and Golgi stack. These data are consistent with the reported visualization of Shiga toxin-horseradish peroxidase conjugates in the ER lumen of butyric-acid treated A431 cells(34) .

Is the relative rate of flux of toxin receptor(s) in the retrograde direction similar to that observed for anterograde transport of cargo? At a concentration of 100 ng/ml, ricin inhibited VSV-G synthesis with a lag of 75 min and a subsequent t of 55 min (Fig. 4, circles). This value is comparable to the rate of anterograde transport of VSV-G to the cell surface in HeLa cells, where VSV-G was found to be transported with a t of 60 min following a 20-min lag as determined by fluorescence-activated cell sorting analysis (Fig. 4, squares). Similar kinetics of intoxication have been observed for Pseudomonas exotoxin A in HeLa cells. (^3)In the case of Shiga toxin, more rapid kinetics have been reported(35) , which were in good agreement with a t of 35 min (2-fold that of ricin) (Fig. 4, triangles) in the HeLa cell line used for the present studies. These values are considerably more rapid than the rate of resialylation of the transferrin receptor in the TGN (t = 2-3 h) (36) or the processing of glycoproteins by cis-Golgi enzymes (t = 4-8 h) following treatment of cells with inhibitors of ER/Golgi-associated mannosidases(37) . These results demonstrate that toxin receptors can move at equivalent rates in the retrograde direction to that of anterograde transported molecules, and suggest a potential for comparable bidirectional membrane flow in the exocytic and endocytic pathways.


Figure 4: Rate of retrograde transport of ricin and SLT-1 is comparable to that of anterograde transport of VSV-G (squares) to the cell surface. Cells were treated with ricin (100 ng/ml) (circles) or SLT-1 (100 ng/ml) (triangles), and VSV-G synthesis and delivery to the cell surface were measured as described under ``Materials and Methods.''



What is the receptor involved in intoxication by ricin-like molecules? Pseudomonas exotoxin A possesses a Lys-Asp-Glu-Leu (KDEL) analog (REDLK) at its carboxyl terminus, which is essential for toxicity(38) . Moreover, adding the ER retrieval sequence KDEL to the carboxyl terminus of ricin significantly increases its cytotoxicity to Vero cells(11) , raising the possibility that the retrograde delivery of extracellular toxins to the ER can be mediated by the KDEL receptor, a protein that is generally considered to be concentrated in pre-Golgi intermediates and the cis-Golgi compartment (reviewed in (39) ). On the other hand, ricin must encounter a galactosylated receptor in the TGN for efficient intoxication. Calreticulin (CaBP3) is an example of a soluble, ``resident'' ER glycoprotein that contains a KDEL retrieval motif, is terminally galactosylated, and has been demonstrated to recirculate between the ER and the trans-Golgi compartments in the liver(40) . Given our results, a reasonable hypothesis is that ricin binds to a molecule such as CaBP3 and is retrieved via the KDEL-mediated pathway. These results suggest that receptors containing a KDEL retrieval signal efficiently recycle from the terminal (trans) Golgi compartments.

Although we have emphasized the interpretation that the effects of these mutants is to disrupt the delivery of a recycling receptor between the ER and TGN, several alternative interpretations are possible. One possibility, albeit a more general interpretation of our specific hypothesis, is that inhibition of anterograde transport leads to the depletion of lipids or other proteins that indirectly effect toxin retrieval from the TGN by globally disrupting the retrograde transport machinery. A second possibility is that disruption of anterograde flow leads to an altered ability of the ER to either receive retrograde transported vesicles or that the lipid composition of the ER is changed, interfering with the ability of toxin to exit to the cytosol. While such alternative conclusions cannot be excluded at this time, we find the effects of the Sar1 mutants, which exclusively block anterograde transport from the ER but do not inhibit transport through the Golgi stack (20) or retrograde retrieval from the Golgi to the ER,^2 offers compelling evidence for the hypothesis that retrograde transport of toxins is mediated by actively recycling receptors. Moreover, when the rate of retrieval of toxin receptors is compared with the markedly reduced rate of retrograde transport of bulk protein based on carbohydrate processing(36, 37) , these data suggest that a significant degree of sorting occurs during retrograde transport through the Golgi stack. This emphasizes the vesicular nature of this pathway and the likelihood for the specific involvement of COP and SNARE complexes.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM42336 (to W. E. B.) and Grant 88/T02035 from the United Kingdom Biotechnology and Biological Sciences Research Council (to J. M. L. and L. M. R.). This is The Scripps Research Institute Manuscript 9193-CB. 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.

§
Both authors contributed equally to this study.

(^1)
The abbreviations used are: TGN, trans-Golgi network; ER, endoplasmic reticulum; SLT-1, Shiga-like toxin-1; PBS, phosphate-buffered saline; VSV, vesicular stomatitis virus; DMEM, Dulbecco's modified Eagle's medium.

(^2)
M. Aridor, T. Rowe, S. Bannykh, and W. E. Balch, submitted for publication.

(^3)
D. Fitzgerald, personal communication.


ACKNOWLEDGEMENTS

We gratefully acknowledge M. Zerial (EMBL) for providing expression plasmids encoding the wild-type and Rab5 mutant, S. Pfeffer (Stanford University) for expression plasmids encoding the wild-type and Rab9 mutant, and S. L. Schmid (Scripps Research Institute) for expression plasmids encoding the wild-type and dynamin element 1 mutants.


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