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Research Article 3503 Evidence that the transport of ricin to the cytoplasm is independent of both Rab6A and COPI Alice Chen, Ramzey J. AbuJarour and Rockford K. Draper* The Molecular and Cell Biology Department, FO31, The University of Texas at Dallas, Box 830688, Richardson, TX 75083-0688, USA *Author for correspondence (e-mail: [email protected]) Accepted 30 April 2003 Journal of Cell Science 116, 3503-3510 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00641 Summary Cholera toxin, Shiga toxin and ricin are examples of protein toxins that require retrograde transport from the Golgi complex into the endoplasmic reticulum (ER) to express their cytotoxic activities and different toxins appear to use different pathways of retrograde transport. Cholera toxin contains the mammalian retrograde targeting signal KDEL and is believed to exploit the coat protein I (COPI) and KDEL receptor-dependent pathway to go from the Golgi complex to the ER. Shiga toxin, however, has no KDEL sequence to specify its inclusion in COPI-coated retrograde vesicles and is believed to use a recently discovered COPI-independent and Rab6Adependent retrograde pathway to enter the ER. Ricin, like Shiga toxin, does not contain a KDEL sequence and is Introduction Cholera toxin, Shiga toxin and ricin are protein toxins that damage mammalian cells by a mechanism that includes four essential events: receptor-mediated endocytosis, retrograde transport into the lumen of the endoplasmic reticulum (ER), passage through the ER membrane into the cytoplasm, and catalytic inactivation of a target substrate in the cytoplasm. The receptor-binding site of a protein toxin is usually within a subunit or domain that is distinct from the subunit or domain that bears the catalytic activity of the toxin. Both cholera toxin and Shiga toxin belong to the AB5 protein toxin family in which the A chain is the enzymatic subunit and each of the five identical B chains bind cell surface receptors. The receptor for cholera toxin is the ganglioside GM1 and the receptor for Shiga toxin is the globotriaosyl ceramide Gb3 (Lingwood, 1996; Spangler, 1992). The A chain of cholera toxin ADP-ribosylates a regulatory G protein and activates adenylate cyclase, which elevates the intracellular cAMP level (Spangler, 1992), whereas the A chain of Shiga toxin arrests protein synthesis by inactivating 28S ribosomal RNA (Endo and Tsurugi, 1987). Ricin has a subunit organization different from the AB5 toxins and contains a single A chain and a single B chain. The B chain binds glycoconjugates that contain galactose residues so that either cell surface glycoproteins or glycolipids that contain galactose can serve as ricin receptors (Sandvig and van Deurs, 2000). The A chain of ricin arrests protein synthesis in target cells by inactivating 28S ribosomal RNA through an enzymatic mechanism identical to that of Shiga toxin (Endo et al., 1987; Endo and Tsurugi, 1987). After binding to cell surface receptors, cholera toxin, Shiga therefore a candidate to use the COPI-independent and Rab6A-dependent pathway of retrograde transport to access the ER. We measured the effect of the GDPrestricted mutant of Rab6A (Rab6A-T27N) on the cytotoxic activity of ricin and found that expressing Rab6A-T27N in cells did not inhibit the cytotoxicity of ricin, suggesting that ricin enters the cytoplasm by a retrograde pathway that does not involve Rab6A. Moreover, ricin still intoxicated cells when Rab6A and COPI were simultaneously inhibited, implying that ricin requires neither Rab6A nor COPI to intoxicate cells. Key words: Ricin, Shiga toxin, Rab6, Retrograde transport, COPI toxin and ricin are endocytosed into vesicles and use pathways of retrograde transport to reach the lumen of the ER (Lencer et al., 1999; Lord and Roberts, 1998; Majoul et al., 1996; Rapak et al., 1997; Sandvig et al., 2002). Once in the ER, there is evidence that the catalytic chains of the toxins pass into the cytoplasm by reverse transport through the Sec61p translocon, the same protein complex used by secretory and membrane proteins to enter the ER from the cytoplasm (Koopmann et al., 2000; Schmitz et al., 2000; Simpson et al., 1999; Wesche et al., 1999). Protein toxins that exploit pathways of retrograde transport from the plasma membrane to the ER have been used as model systems to characterize the transport pathways. The best understood pathway of retrograde transport involves coat protein I (COPI), a large protein complex that is primarily found on vesicles budding from Golgi membranes (Cosson and Letourneur, 1997; Lippincott-Schwartz et al., 1998; Rothman and Wieland, 1996). The major component of COPI is coatomer, a cytoplasmic protein complex that contains seven subunits (α-, β-, β′-, γ-, δ-, ε- and ζ-COP) (Scales et al., 2000). Recruitment of COPI onto membranes depends on the activity of ADP ribosylation factor 1 (ARF1), a small GTPase protein, and its effectors (Aoe et al., 1997; Aoe et al., 1998). Together, coatomer and ARF1 constitute COPI. One function of COPI is to select proteins in the Golgi apparatus for retrograde transport to the ER. Proteins destined for Golgi to ER transport carry amino acid signals that allow them to interact, either directly or indirectly, with COPI and be included in vesicles that bud at sites coated by COPI. One well-characterized amino acid signal specifying retrograde 3504 Journal of Cell Science 116 (17) transport is the KDEL sequence present on the C-terminus of soluble proteins that are residents of the ER. These proteins normally function within the ER, but if they escape to the Golgi, they are returned to the ER via COPI-coated vesicles. A major insight into the retrograde transport of protein toxins was that some toxins, such as cholera toxin, contain the KDEL sequence that allows them to be transported in COPI-coated vesicles from the Golgi to the ER (Lencer et al., 1995; Majoul et al., 1996). Although cholera toxin contains a KDEL sequence, other toxins that require retrograde transport to reach the ER, such as ricin and Shiga toxin, do not contain this amino acid motif. In the absence of a known retrograde signal on these toxins, the mechanism by which they access the ER had been unknown. Recently, however, a second pathway of retrograde transport that is independent of COPI (Girod et al., 1999) was discovered, based in part on the study of Shiga toxin. This work suggests that toxins bearing KDEL motifs enter the ER by the classical COPI-dependent pathway and those toxins without KDEL motifs engage the COPI-independent pathway. The COPI-independent pathway appears to be regulated by the small GTP binding protein Rab6A (Girod et al., 1999; White et al., 1999). Rab6A is one of two Rab6 isoforms ubiquitously expressed in mammalian cells and is associated with medial and trans-Golgi cisternae as well as the trans Golgi Network (TGN) (Antony et al., 1992; Echard et al., 2000). Evidence that Rab6A functions in COPI-independent retrograde transport includes the observation that expression in cells of a GDP-restricted form of Rab6A (Rab6A-T27N) inhibits the transport of Shiga toxin to the ER and reduces the cytotoxic activity of Shiga toxin (Girod et al., 1999). It is not clear, however, whether other toxins lacking a KDEL signal also use the Rab6A-dependent pathway to enter the ER. We measured the effect of the Rab6A-T27N mutant on the cytotoxic activity of ricin, which has no KDEL sequence. We also studied the effect of expressing Rab6A-T27N on the action of ricin under circumstances in which the COPI-dependent pathway of retrograde transport was inhibited. Our data suggests that the transport of ricin to the ER is independent of both Rab6A and COPI. Materials and Methods Materials Ricin, bovine serum albumin and rhodamine-labeled secondary antibodies were from Sigma Chemical Company (St. Louis, MO, USA). Cholera toxin was from Calbiochem (La Jolla, CA, USA). Rabbit antiserum to TGN-38 was generously provided by Dr S. Milgram (The University of North Carolina, Chapel Hill, NC, USA). Shiga toxin was purchased from Toxin Technology (Sarasota, FL, USA). cDNA encoding human Rab6A was provided by Dr W. Maltese (Medical College of Ohio, Toledo, OH, USA). Rab6A DNA was digested with EcoRI and BamHI and inserted into the vector pEGFPC2 (Clontech Laboratories, Palo Alto, CA, USA) to generate the Rab6A-GFP construct. A plasmid containing Rab6A-T27N was kindly provided by Dr B. Goud (Institut Curie, Paris, France). Tran 35S-label was from ICN Radiochemical (Irvine, CA, USA). Dulbecco’s modified eagle’s medium (DMEM) was from Irvine Scientific (Santa Ana, CA, USA). Fetal Bovine serum was from HyClone (Logan, UT, USA). Intracellular cAMP was measured with a kit purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Cell culture LdlF cells, originally derived from Chinese hamster ovary (CHO) K1 cells (Guo et al., 1996; Guo et al., 1994; Hobbie et al., 1994) were provided by Dr Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA, USA). Vero cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were routinely grown in DMEM supplemented with 10 mM HEPES and 5% fetal bovine serum in incubators with 90% air and 10% CO2 at either 34°C, 37°C or 39.5°C as required. Expression of Rab6A-T27N Cells were transfected with expression plasmids encoding either wildtype Rab6A or mutant Rab6A (Rab6A-T27N) using LipofectAMINE 2000 (Life Technologies, Inc., Rockville, MD, USA) following the protocol provided by the vendor. In brief, cells were plated in 24-well plates at a density of 2×105 cells per well one day before a transfection. To transfect cells, 1 µg of plasmid DNA was added to 50 µl of the transfection medium (serum-free DMEM) and 2 µl of LipofectAMINE 2000 was added to another 50 µl of transfection medium for 5 minutes incubation at room temperature. The samples containing the plasmid and the LipofectAMINE 2000 were mixed and incubated at room temperature for another 30 minutes. Cells were washed with phosphate-buffered saline to remove residual serum and 500 µl of serum-free DMEM was added to each well. Mixed transfection medium (100 µl) containing plasmid and LipofectAMINE 2000 was then added to each well and incubated for 18 to 24 hours to allow expression of the gene encoded on the plasmid. To measure the efficiency of transfection, an additional set of cells were prepared at the same time for immunofluorescence microscopy and stained with a primary antibody to the protein of interest. The percentage of cells in several fields that were positive for the protein of interest was the transfection efficiency and was between 60% and 75% of the total cells. Analysis of influenza virus HA protein transport The effect of Rab6A-T27N on the transport of influenza virus hemagglutinin protein (HA) from the ER to the cell surface was measured by incorporation of radioactivity from Tran 35S-label into HA as previously described (Hu et al., 1999). In brief, cells were transfected with plasmid encoding wild-type Rab6A or the mutant Rab6A-T27N one day before an experiment. Vero cells were infected with influenza virus (X31) at 37°C for 30 minutes, washed and incubated for a further 3.5 hours. LdlF cells were infected with influenza virus (Japan) at 34°C for one hour, washed and incubated for 4 hours. Subsequently, all cells were incubated for 15 minutes with assay medium containing 100 µCi/ml of Tran 35S-label. The radioactive medium was then removed and 0.2 ml of fresh assay medium was added for further incubation time as indicated in the Figures and Tables. To assess the appearance of HA on the cell surface, cells were treated with extracellular trypsin, which cleaves surface HA into two fragments. Radiolabeled cells were rinsed once with PBS and treated on ice with phosphate-buffered saline containing 100 µg/ml TPCKtrypsin for 30 minutes. Soybean trypsin inhibitor (100 µg/ml) was then added for 10 minutes before the cells were lysed with lysis buffer. HA (X31) was immunoprecipitated overnight with mouse monoclonal antibody FC125 while HA (Japan) immunoprecipitated with polyclonal anti-HA. The immunoprecipitates were incubated at 65°C for 30 minutes in 50 mM sodium citrate, pH 5.5, containing 0.1% SDS, and each sample was mixed with sample buffer before electrophoresis in a 12% SDS-polyacrylamide gel. Radiolabeled protein bands were scanned by PhosphorImaging with the STORM system and quantitated by ImageQuant 5.0 (Molecular Dynamics, Sunnyvale, CA, USA). HA0 is intact HA, HA1 is the large trypsin fragment and HA2 is the small trypsin fragment. The following GDP-restricted Rab6A does not inhibit ricin formula was used to determine the fraction of HA proteins that were sensitive to trypsin and therefore on the cell surface: Trypsin-sensitive fraction (%)=100×[(HA1 + HA2)/(HA0 + HA1 + HA2)]. Three independent experiments were performed with Vero cells and the results are presented as the mean±the standard error of the mean (s.e.m.). Protein synthesis assay Protein synthesis was measured by the incorporation of radioactivity from Tran 35S-label into acid insoluble protein essentially as described previously (Hu et al., 1999). Cells were transfected as indicated in Table legends and a toxin was added at different concentrations for the indicated times. Tran 35S-label (1 µCi/ml) was added for 30 minutes and the cells were washed, lysed, and the lysate spotted within one-inch squares defined by gridlines drawn on filter paper. The filter paper was incubated in 5% trichloroacetic acid containing 0.5 mg/ml methionine for 30 minutes at room temperature, washed twice for 5 minutes in 100% ethanol, and dried. Radioactivity within each square of the grid was measured and quantitated with a PhosphorImager from Molecular Dynamics. The IC50 value is defined as the concentration of toxin required to inhibit protein synthesis by 50%. Controls in these experiments were either cells carried through the transfection procedure without plasmid (mock-transfected cells) or cells transfected with plasmid encoding wild-type Rab6A, as indicated in the Figure legends. There was no difference in the response to toxins of mock-transfected cells and cells expressing wild-type Rab6A. For experiments done three or more times, the IC50±s.e.m. is presented. Kinetics of protein synthesis inhibition by ricin The rate of ricin intoxication was measured by assessing the incorporation of radioactivity from Tran 35S-label into acid insoluble protein as described previously (Bau and Draper, 1993) with modifications. Cells were transfected as indicated in Fig. 3 for 18 hours before an experiment. On the day of the experiment, the cells were chilled at 0°C and incubated with DMEM (lacking methionine) in the presence of ricin (100 µg/ml) for an hour to allow toxin binding to cell surface receptors. Intoxication was initiated by replacing the cold medium with pre-warmed assay medium and incubation was continued at 37°C. Tran 35S-label (10 µCi/ml) was added for 10 minutes at desired time points. The cells were washed, lysed and radioactivity measured as described in the preceding section. 3505 were incubated with fixed and permeabilized cells for 30 minutes at room temperature, washed as in the previous sentence, followed by addition of rhodamine-labeled secondary antibody and a final washing. Coverslips were mounted and viewed with a Nikon TE300 microscope equipped with epi-illuminated fluorescence and a 60X lens (NA=1.4) as previously described (Chen et al., 2002). Images were obtained with a MicroMax digital camera (Princeton Instruments, Trenton, NJ, USA). Pixel intensities were scaled and images were assembled in Adobe Photoshop 5.5 (Adobe Systems, Inc., San Jose, CA, USA). Results The effect of Rab6A-T27N on secretion and on the cytotoxicity of Shiga toxin and cholera toxin Expression in HeLa cells of the GDP-restricted form of Rab6A (Rab6A-T27N) delays the secretion of the influenza virus HA protein (Martinez et al., 1994) and partially inhibits the cytotoxic activity of Shiga toxin (White et al., 1999). To verify that expression of Rab6A-T27N had similar consequences in Vero cells, we measured the effect of expressing Rab6A-T27N on both HA secretion and on the sensitivity of the cells to Shiga toxin. Transfected and control cells were infected with influenza virus, pulsed with radioactive methionine, and incubated for an hour to allow transport of HA to the cell surface. Cells expressing Rab6A-T27N transported less HA to the cell surface in 60 minutes than control cells (Fig. 1). When the results of three independent experiments were quantitated, 50%±5 of the HA was on the surface of control cells within 60 minutes compared to 26%±6 for cells expressing Rab6AT27N. These results confirm that Rab6A-T27N impairs secretion of HA in Vero cells similar to that reported for HeLa cells (Martinez et al., 1994). The effect of Rab6A-T27N on the sensitivity of cells to Shiga toxin was assessed by measuring protein synthesis in transfected and control Vero cells as a function of toxin concentration. Cells transfected with the plasmid encoding Rab6A-T27N displayed moderate resistance to Shiga toxin Cholera toxin assays The effect of cholera toxin on cAMP levels in Vero cells and ldlF cells was directly measured. Cells in 24-well culture plates were transfected with a plasmid containing the protein of interest and incubated for 18 hours to express the protein. Cells were then incubated with or without 1 µg/ml cholera toxin as indicated in the Table legends. Cells were lysed and intracellular cAMP levels were directly measured with an immunological assay according to instructions supplied with the Amersham Pharmacia Biotech assay kit. Immunofluorescence microscopy To transiently express Rab6A-GFP, ldlF cells were transfected with plasmid using LipofectAMINE PLUS (Life Technologies, Rockville, MD, USA) following the protocol provided by the vendor. After 48 hours at 34°C, half the samples were shifted to the restrictive temperature of 39.5°C. For indirect immunofluorescence, cells were fixed in 4% paraformaldehyde and permeabilized by 0.2% Saponin for 10 minutes. Samples were then washed 3 times with phosphatebuffered saline and incubated with phosphate-buffered saline containing 1% BSA for 10 minutes. Primary antibodies to TGN-38 Fig. 1. The effect of Rab6A-T27N on the transport of HA to the plasma membrane in Vero cells. Vero cells were transfected with or without Rab6A-T27N for 18 hours, infected with influenza virus, radiolabeled and then chased for 60 minutes as described in Materials and Methods. After trypsin treatment, the cells were lysed and HA proteins were immunoprecipitated and resolved in a 12% SDS-polyacrylamide gel. Transport to the plasma membrane was determined by trypsin sensitivity as described in Materials and Methods. HA0 is intact HA whereas HA1 and HA2 are the large and small trypsin fragments, respectively. 3506 Journal of Cell Science 116 (17) Table 1. The effect of Rab6A-T27N on the action of cholera toxin in Vero cells cAMP (fmol well–1) Plasmid None Rab6A-T27N − cholera toxin + cholera toxin 483 313 10 278 9 791 Cells were either mock transfected or transfected with the plasmid encoding Rab6A-T27N for 18 hours. Cells were then incubated with or without 1 µg ml–1 cholera toxin on ice for one hour to allow toxin binding. Samples were shifted to 37°C for another hour to allow time for the toxin to act. Cells were lysed, and intracellular cAMP levels were directly measured as described in Materials and Methods. Values are the average from two independent experiments. Shiga toxin, but does not affect the pathway of retrograde transport used by cholera toxin. Fig. 2. The effect of Rab6A-T27N on the sensitivity of cells to Shiga toxin and ricin. Vero cells were either mock transfected (䊊) or transfected with a plasmid encoding mutant Rab6A-T27N (䊐) for 18 hours and treated with various concentrations of Shiga toxin (A) or ricin (B) for 2.5 hours. Tran 35S-label was added for 30 minutes and the incorporation of radioactivity into acid-insoluble material was determined as described in Materials and Methods. Each point is the average of three independent experiments. The effect of Rab6A-T27N on the cytotoxicity of ricin The inhibition of protein synthesis as a function of ricin concentration for control cells and cells expressing Rab6AT27N was nearly identical (Fig. 2B), yielding IC50 values for ricin of 15±7 ng/ml and 22±4 ng/ml for control cells and cells expressing Rab6A-T27N, respectively. To further characterize the effect of Rab6A-T27N on the entry of ricin into Vero cells, we compared the rate at which ricin is transported from the cell surface to the cytoplasm in transfected and control cells. Cells were chilled, incubated with high concentrations of ricin to saturate cell surface receptors, returned to 37°C, and protein synthesis was assessed at different times. A plot of the logarithm of the inhibition of protein synthesis versus time yields a straight line in this type of analysis and extrapolation of the graph to no inhibition of protein synthesis provides the minimum time required for the first burst of toxin molecules to reach the cytoplasm (Neville and Hudson, 1986). The time required for ricin to reach the cytoplasm of both control cells and cells expressing Rab6A-T27N was indistinguishable, approximately 30 minutes (Fig. 3). Thus, expression of Rab6A-T27N had no significant effect on either the IC50 for ricin or the time required for ricin to enter the cytoplasm and inhibit protein synthesis. (Fig. 2A). The average concentration of Shiga toxin required to reduce protein synthesis by 50% (IC50) in three independent experiments was 9±2 ng/ml for control cells and 32±2 ng/ml for cells expressing Rab6A-T27N. Thus, expressing the GDPrestricted form of Rab6A partially inhibited the cytotoxicity of Shiga toxin in Vero cells, consistent with previous results in HeLa cells (White et al., 1999). Cholera toxin is believed to access the lumen of the ER by a pathway of retrograde transport that is independent of Rab6A; therefore, expressing Rab6A-T27N is not expected to interfere with the action of cholera toxin. To test this, cells making Rab6A-T27N and control cells were incubated with or without 1 µg/ml of cholera toxin and the level of cytoplasmic cAMP was measured. There was no discernible influence of Rab6-T27N on the ability of cholera toxin to elevate cAMP levels (Table 1) in Vero cells. Altogether, the results in this section suggest that Rab6A-T27N impairs secretion, impairs the COPI-independent retrograde transport pathway used by The effect of simultaneous inhibition of COPI and Rab6A on the action of ricin and cholera toxin We recently noted that ldlF cells, a strain of CHO cells that carries a temperature-sensitive mutation in the ε subunit of COPI, were sensitive to ricin at the restrictive temperature (Chen et al., 2002). This suggested that ricin reaches the cytoplasm by a pathway that does not require ε-COP, such as the Rab6A-dependent pathway used by Shiga toxin. However, the results with Rab6A-T27N in the previous section suggest that ricin still reaches the cytoplasm when the Rab6Adependent pathway is impaired. One explanation for this is that ricin may use either the COPI-dependent or the Rab6Adependent pathway, and when one is blocked, the toxin can still access the cytoplasm by the other pathway. To test this, we measured sensitivity of cells to ricin under conditions in which both the COPI-dependent and Rab6A-dependent pathways should be inhibited by expressing Rab6A-T27N in ldlF cells at the restrictive temperature. GDP-restricted Rab6A does not inhibit ricin 3507 Table 2. The effect of Rab6A-T27N on the action of ricin in ldlF cells at 34°C and 39.5°C Plasmid Rab6A Rab6A-T27N Rab6A Rab6A-T27N °C IC50 (ng ml–1) 34 34 39.5 39.5 20 14 11 8 Cells were transiently transfected with plasmids containing either wild-type or mutant Rab6A for 18 hours at 34°C. One-half of the samples were shifted to 39.5°C and incubated for another 18 hours. The cells were exposed to different concentrations of ricin for 4.5 hours. Tran 35S label was added for the last 30 minutes of the incubation and the incorporation of radioactivity into acid-insoluble material was determined as described in Materials and Methods. Values are the average of two independent experiments. Fig. 3. The effect of Rab6A-T27N on the rate at which ricin first enters the cytoplasm. Mock-transfected control cells (䊊) or cells transfected with Rab6A-T27N (䊐) for 18 hours were chilled and incubated with 100 µg/ml of ricin for an hour to bind the toxin to cell surface receptors. The cells were raised to 37°C by addition of warm medium and protein synthesis was assessed at the indicated times as described in Materials and Methods. The solid (control) and broken (transfected with Rab6A-T27N) lines were fitted to the data points by the method of least squares. We first characterized the expression of wild-type Rab6A in ldlF cells at permissive and restrictive temperatures by immunofluorescence microscopy. Rab6A was imaged in cells transfected with a plasmid expressing Rab6A-GFP followed by staining for TGN-38 to co-visualize the TGN. In ldlF cells at the permissive temperature, TGN-38 co-localized closely with Rab6A-GFP in transfected cells (Fig. 4A,B). At the high Fig. 4. The distribution of TGN-38 and Rab6A-GFP in ldlF cells. Cells were transfected with plasmid DNA encoding Rab6A-GFP and incubated for 48 hours at 34°C. Cells were left at 34°C (A and B) or shifted to 39.5°C for 6 hours (C and D). The cells were then fixed and stained with anti-TGN-38 followed by a rhodamine-labeled secondary antibody. (A) and (C) show rhodamine-labeled anti-TGN38 and B and D are direct visualization of GFP in the fields shown in A and C, respectively. The arrows in C and D show examples of structures that label with both TGN-38 and Rab6A-GFP in ldlF cells at the high temperature. Scale bar in D: 25 µm. temperature, TGN-38 was present in a variety of perinuclear structures (Fig. 4C), suggesting that TGN-like membranes persisted in the mutant cells at the high temperature, consistent with previous results that similar structures containing γadaptin were present in the ldlF cells at high temperature (Chen et al., 2002). The structures that contained TGN38 in ldlF cells at high temperature also stained for Rab6A-GFP (Fig. 4D). These data demonstrate that wild-type Rab6A is expressed in transfected ldlF cells and that it associates with perinuclear membranes that also bind TGN-38 at both permissive and restrictive temperatures. We also characterized the effect of Rab6A-T27N on secretion in ldlF cells to ensure that the dominant-inhibitory mutant was having the expected effect on secretory function. LdlF cells were transfected with plasmid encoding Rab6A-T27N, infected with influenza virus at 34°C, labeled with Tran 35S-label, and the extent of HA on the cell surface was compared to cells that had not been transfected. Transfected cells had a 46% reduction in the amount of HA secreted compared to control cells, consistent with the effects of Rab6A-T27N on retarding secretion in HeLa and Vero cells. The effect of Rab6A-T27N on the sensitivity of ldlF cells to ricin is shown in Table 2. The IC50 for ricin at the permissive temperature of 34°C in the absence of Rab6A-T27N was 20 ng/ml and in the presence of Rab6A-T27N was 14 ng/ml. These control data support the results with Vero cells that expression of Rab6A-T27N does not inhibit the access of ricin to the cytoplasm. At the restrictive temperature, the IC50 values for ricin in the absence and presence of Rab6A-T27N were 11 ng/ml and 8 ng/ml, respectively, suggesting that ricin efficiently reaches the cytoplasm even when the functions of both COPI and Rab6A are impaired. Similar experiments were done with cholera toxin in the ldlF cell system (Table 3). Expression of Rab6A-T27N at 34°C did not affect the response of ldlF cells to cholera toxin. The response of the cells to cholera toxin is partially reduced by incubation at 39.5°C, as previously reported (Chen et al., 2002), and expression of Rab6A-T27N did not affect the residual response. Thus, cholera toxin still reaches the cytosol of ldlF cells regardless of whether Rab6A function is impaired. The effects of Shiga toxin could not be tested in this system because neither LdlF cells nor their parental CHO cell line are sensitive to Shiga toxin. Discussion Three different controls were done to verify the effects of 3508 Journal of Cell Science 116 (17) Table 3. The effect of Rab6A-T27N on the action of cholera toxin in ldlF cells at 34°C and 39.5°C Clathrin-independent Endocytosis Clathrin-dependent Endocytosis cAMP (fmol well–1) Plasmid °C − cholera toxin + cholera toxin Rab6A Rab6A-T27N Rab6A Rab6A-T27N 34 34 39.5 39.5 506 750 260 172 11 263 11 966 3 629 3 382 Cells were transiently transfected with plasmids containing either wild-type Rab6A or Rab6A-T27N for 18 hours at the permissive temperature. One-half of the samples were shifted to 39.5°C and incubated for another 6 hours before addition of 1 µg ml–1 cholera toxin for 18 hours. Cells were lysed, and intracellular cAMP levels were directly measured as described in Materials and Methods. Values are the average from two independent experiments. Caveosomes Early Endosomes Rab6A' Recycling Endosomes Rab7 Late Endosomes Rab6A' Rab11 Rab9 TGN expressing Rab6A-T27N on membrane traffic in Vero cells. First, the secretion of the influenza virus HA protein was monitored in cells transfected with a plasmid encoding Rab6A-T27N and an inhibitory effect on HA protein secretion was observed, as noted by Martinez et al. (Martinez et al., 1994). Second, to confirm that Rab6A-T27N interfered with the COPI-independent retrograde transport pathway, the sensitivity of Vero cells to Shiga toxin was measured after expression of Rab6A-T27N in the cells. The cytotoxicity of Shiga toxin was partially inhibited, replicating results observed with Rab6A-T27N in Hela cells (Girod et al., 1999). Finally, we measured the effect of Rab6A-T27N on cholera toxin, a toxin that contains a KDEL sequence and which should not need the Rab6A-dependent pathway to intoxicate cells. Expressing Rab6A-T27N did not inhibit the action of cholera toxin in Vero cells. These controls are evidence that expressing Rab6A-T27N interfered with functions believed to depend on Rab6A, but not on functions independent of Rab6A. The effect of Rab6A-T27N on the cytotoxicity of ricin was then assessed and the major observation here is that neither the IC50 for ricin, nor the rate at which ricin intoxicated cells, was affected in cells expressing the GDP-restricted derivative of Rab6A. There are two isoforms of Rab6 in mammalian cells generated by alternative splicing, Rab6A and Rab6A′ (also called Rab6C), that differ by only three amino acids (Echard et al., 2000). Interestingly, there is evidence that the two isoforms have different functions. Rab6A is implicated in regulating the COPI-independent retrograde transport pathway from the Golgi complex to the ER because Rab6A-T27N inhibits this pathway (Girod et al., 1999; White et al., 1999). Our observation that Rab6A-T27N failed to interfere with the action of ricin in either Vero cells or ldlF cells argues that ricin does not require the COPI-independent and Rab6A-dependent pathway used by Shiga toxin to go from the Golgi to the ER. Rab6A′ is believed to participate in transport from early/recycling endosomes to the TGN because Rab6A′-T27N, and antibodies to Rab6A′, inhibit this pathway (Mallard et al., 2002). It is difficult, however, to know whether a GDPrestricted mutant interferes with either Rab6A or Rab6A′ because, with the exception of Rabkinesin 6, all known Rab6 binding proteins interact with both Rab6A and Rab6A′ (Echard et al., 2000). Consequently, the GDP-restricted mutant of either one of the two Rab6 isoforms could sequester essential factors needed by the other isoform and it has been found that expressing Rab6A-T27N in HeLa cells inhibited the transport of Shiga toxin from endosomes to the TGN, a pathway believed Golgi COPI-independent Rab6A-dependent COPI-dependent Rab6A-independent Endoplasmic Reticulum Fig. 5. Rab proteins involved in retrograde transport from endosomes to the ER. Toxins enter cells either by clathrin-independent or clathrin-dependent endocytosis. Initial pathways of uptake converge on early endosomes, except for the caveosome pathway which apparently bypasses the typical endosomal system in transporting material to the ER. One pathway from early endosomes to the TGN is via late endosomes that use Rab7 and Rab9 in sequential steps. Another pathway to the TGN is via recycling endosomes and involves Rab6A′ and Rab11 in different steps. Rab6A′ may also participate in the direct transport of material from early endosomes to the TGN. Two pathways are proposed to transport material from the TGN-Golgi complex to the ER, the COPI-dependent pathway that is regulated by Rab6A and the COPI-independent pathway that is not believed to require Rab6A. to require Rab6A′, not Rab6A (Mallard et al., 2002). Thus, our data is consistent with the possibility that ricin does not require the early/recycling endosome-to-TGN pathway controlled by Rab6A′. This prospect has interesting implications because it has previously been shown that ricin transport to the Golgi is independent of Rab9 and Rab11 (Iversen et al., 2001). Because the only known pathways from endosomes to the TGN involve either Rab9 or Rab11 or Rab6A′, the pathway ricin uses to reach the TGN is unidentified (see Fig. 5 for an overview of Rab proteins and pathways of retrograde membrane traffic). What Rab6A-independent pathway of retrograde transport does ricin use to go from the Golgi complex to the ER? It is conceivable that ricin uses the same COPI-independent pathway as Shiga toxin, but that Rab6A is not essential for ricin to move through this pathway. This would imply that some ligands, such as Shiga toxin, require Rab6A to engage this pathway whereas others, perhaps ricin, do not. A second possibility is that ricin uses the established COPI-dependent pathway upon binding to galactose residues of a carrier glycoprotein that bears the KDEL motif specifying inclusion in COPI-coated vesicles. However, interfering with COPI function in two different ways fails to protect cells from ricin (Chen et al., 2002), suggesting that the COPI-dependent pathway is not essential for ricin to access the cytoplasm. It is GDP-restricted Rab6A does not inhibit ricin also conceivable that ricin can use more than one pathway of retrograde transport and when one pathway is inhibited, another is still available. To test this, we simultaneously inhibited COPI and Rab6A by raising the temperature in ldlF cells that were expressing Rab6A-T27N. Ricin still intoxicated the cells when both pathways were impaired, a significant observation because it suggests that neither the COPIdependent nor the COPI-independent pathway is essential for the transport of ricin to the cytoplasm. Interestingly, cholera toxin still had residual activity when both pathways were impaired, suggesting the availability of a pathway independent of COPI and Rab6A for this toxin as well. It is also possible that ricin reaches the ER by a caveolar pathway. For example, the transit of SV-40 virus to the ER via a cytoplasmic organelle termed the ‘caveosome’ has recently been described (Pelkmans et al., 2001; Pelkmans et al., 2002). In addition, caveolin-positive endosomes have been implicated in the delivery of cholera toxin to the Golgi complex from which it may access the ER (Nichols, 2002). Ricin transport is complicated by the likelihood that multiple pathways participate in the uptake of this toxin and it is difficult to identify pathways that directly deliver ricin to the ER. To address this, we are continuing to study conditions that simultaneously inhibit two or more transport pathways in the event that this will isolate the most important pathways required for ricin to reach the ER. This work partially fulfills the requirement for the Ph.D. degree for A. Chen. We thank Dr M. Krieger for providing ldlF cells, Dr S. Milgram for anti-TGN-38, Dr W. Maltese for cDNA encoding Rab6A, and Dr B. Goud for cDNA encoding Rab6A-T27N. We thank C. Mikoryak for comments on the manuscript. We are also indebted to Dr M. Roth for assistance with influenza virus. 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