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Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL.

Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin-containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GM1 at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced Cl- secretion. At T1/2, dose dependencies for K(R)DEL-mutant toxins were increased > or = 10-fold. KDEL-mutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction. KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.

placing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a C1-secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced C1secretion. At T1/2, dose dependencies for K(R)DELmutant toxins were increased ~>10-fold. KDELmutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction, KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.
ETROGRADE transport through Golgi cisternae has been shown to occur for soluble and membrane proteins of the ER (49) and for certain protein toxins (6,55). Targeting of soluble ER and some type II membrane proteins in this pathway depends on the COOH-terminal sorting signal Lys-Asp-Glu-Leu (KDEL or HDEL toxin, LT) also contain respectively the KDEL or the equally potent sorting signal RDEL at the C-terminus of their enzymatically active A subunits (59,60). CT and LT are structurally, immunologically, and functionally homologous (reviewed in reference 61). Both toxins act directly on intestinal epithelia to elicit massive secretory diarrhea by activating cAMP-dependent signal transduction pathways. Both CT and LT are comprised of five identical B subunits that bind with high affinity to ganglioside GM1 at the cell surface (7,17,25), and a single enzymatic A subunit comprised of two peptides linked by a disulfide bond (59,66). After reduction of the A subunit, the enzymatically active At-peptide (~22 kD) acts inside the cell to activate adenylate cyclase by catalyzing the ADP-ribosylation of the heterotrimeric GTPase Gsct (5,39,40). The A2-peptide (~5 kD) contains the COOH-terminal K(R)DEL motif that protrudes unhindered below the plane formed by the pentameric B subunit on the side which binds GMI at the cell surface (36,59,60).
The presence of K(R)DEL at such a site on both CT and LT raises the distinct possibility that both toxins require direct interactions with endogenous KDEL-receptors for biologic activity (50). The KDEL-receptor is a seven-transmembrane domain protein localized predominantly to the intermediate compartment and Golgi cisternae with a graded distribution from cis to trans (16,63). The KDEL-receptor mediates retrograde transport of KDEL-containing proteins from multiple sites within Golgi cisternae including trans-Golgi (8,20,31,37,52).
The process by which CT elicits a response in polarized and nonpolarized cells takes 20-40 min (termed the "lag" phase) (11,28) and involves movement of CT through multiple intracellular compartments. Binding of CT to the cell surface is followed by endocytosis and subsequent transport of toxin containing membranes to a functionally defined intracellular compartment(s) where reduction and membrane translocation of the A subunit occurs (27,45). The anatomical compartment of A subunit translocation, however, remains unknown though available evidence suggests this event may occur in Golgi cisternae. For example, CT-labeled horseradish peroxidase or gold particles have been shown to move from the cell surface via endosomes to trans-Golgi (23), and recently, a small fraction of internalized CT B subunit has been localized within cis elements of Golgi cisternae (56). These data provide direct evidence that CT can move retrograde in the secretory pathway. Neither study, however, provides evidence that transit through Golgi is physiologically relevant to the cellular response.
The lag phase ends with activation of adenylate cyclase by toxin-catalyzed ADP-ribosylation of Gsa. Activation of adenylate cyclase occurs by movement of the reduced and translocated Al-peptide (or possibly another messenger) back to the plasma membrane. The events that follow reduction (and presumably translocation [24]) of the A subunit are not necessarily diffusion limited. In polarized T84 cells, for example, signal transduction by the reduced Al-peptide is completely inhibited late in the lag phase by lowering incubation temperatures from 37 to 15°C (28). These and other data (13,14,69) suggest that the At-peptide may not break free of the membrane after translocation. We have recently found that after translocation the Arpeptide may not dissociate from membrane vesicles containing the B subunit. Both subunits may move together via vesicular carriers from the site of translocation to adenylate cyclase on the basolateral membrane (29).
Our aim in the present study was to define intermediary compartments through which CT and LT must physiologically traffic to elicit a cellular response. We examined whether the COOH-terminal K(R)DEL motif of the A2-peptide may play a role in toxin action. If K(R)DEL were required for normal CT or LT signal transduction, retrograde movement of CT/LT from Golgi to ER would be implied. Recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine was made. Toxin action was examined in the human intestinal cell line T84, a polarized cell model of intestinal epithelia and the natural target of CT in nature. T84 cells grown on permeable supports form confluent monolayers of columnar epithelia that exhibit high transepithelial resistance, polarized apical and basolateral membranes, and a cAMP-regulated C1-secretory pathway analogous to that found in intact intestine (1). Activation of adenylate cyclase in T84 cells by intoxication with CT or LT elicits a strong CI-secretory response that can be detected using standard electrophysiologic techniques. Our data provide strong evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.

Cell Culture
T84 cells obtained from ATCC were cultured and passaged as previously described (9) in equal parts of DME (1 g/L D-glucose) and Ham's F-12 Nutrient mixture, supplemented with 5% newborn calf serum, 15 mM Hepes, 14 mM NaHCO3, 40 mg/L penicillin, 8 mg/L ampicillin, and 0.90 mg/L streptomycin. Cells were seeded at confluent density onto 0.33-or 5-cm 2 Transwell inserts (Costar Corp., Cambridge, MA) coated with a dilute collagen solution as previously described (33). Transepithelial resistances attained stable levels (>1,000 ohms cm 2) after 7 d. The development of high transepithelial resistance correlated with the formation of confluent monolayers with well-developed tight junctions as assessed by morphological analysis (32), and with the ability of monolayers to secrete CI-. Cells from passages 71-84 were utilized for these experiments. directed mutagenesis kit (Amersham International, Arlington Heights, IL) using single-stranded pTRH29 as a template and a synthetic oligonucleotide (5'-TATTCAT__ACTTCATCC-3', from the Microanalytical Facility, IAPGR, Cambridge Research Station, UK) as the mutagenic primer. The correct Leu to Val substitution was confirmed by dideoxy sequencing using Sequenase I1 (United States Biochemical Corp., Cleveland, OH) and the resultant pIasmid designated pTRH73. The clone producing the CT variant with LEDERAS substituted for KDEL at the COOH terminus of the A subunit was constructed in two steps. First the Lys 237 codon AAG (italicized letters in the sequence GAATI'AAG) in pMGJ60 was changed by oligonucleotide-directed mutagenesis to CAG, thereby creating the EcoR1 restriction site GAATTC in the resulting plasmid pMGJ67 (22). Next, a PvuII-EcoRI fragment of pMGJ67 that encodes the IPTGinducible, truncated ctxA gene was subcloned into PvuII-EcoRI cut pBR322 to generate pMGJ117, and the predicted fusion joint was confirmed by dideoxy sequencing. Fusion of ctxA to vector-derived sequences in pMGJll7 substitutes LEDERAS for KDEL at the COOH terminus of the A subunit of CT, in a manner equivalent to the LEDERAS variant of LT constructed by Sanchez et al. (54). To produce holotoxin with the LEDERAS variant of CT-A, pMGJ117 was transformed into E. coli TX1 (Stratagene Inc., La Jolla, CA) containing pMGJ85, a compatible plasmid clone that expresses ctxB from the IPTG-inducible promoter of pK184 (21).

Preparation of Periplasmic Extracts Containing Recombinant Wild-type and
Mutant CTand LT. E. coli XL1-Blue (fecAl lac-endA1 gyrA96 thi hsdR17 supE44 relA1 IF' proAB lacIq lacZAM15 Tnl0]) (4) or TX1 harboring plasmids encoding wild-type or mutant CT or LT were inoculated from stocks into 200 ml Luria-Bertani medium (L-broth) (53) supplemented with 200 ~g/ml ampicillin and 10 p.g/ml tetracycline. The cells were grown at 37°C on a rotary shaker. When the culture had reached an optical density of 0.2 (600 nm), isopropyl-l-thio-13-o-galactopyranoside (0.5 mM final) was added and the cells cultured for an additional 2 h. The cells were then collected by centrifugation (6,000 rpm, 4°C for 15 rain) and washed in 4 ml of ice-cold PBS (NaC1 140 raM, Na2HPO3/NaH2PO3 10 mM, pH 7.2) and then resuspended in 8 ml of ice-cold 0.3 M sucrose, 0.1 M Na2HPO3/ NaH2PO3, pH 7.6. EDTA (5 mM) and lysozyme (10 p.g/ml) were added and the cells kept on ice for an additional 15 min with occasional agitation. The treated cells were centrifuged (12,000 rpm at 4°C for 5 min) and the supernatant containing the periplasmic extract removed, dialyzed overnight against two changes of HBSS (1:1,000 vol/vol), separated into 20-50-~l aliquots, flash frozen, and stored at -80°C until required.

Electrophysiology
For electrophysiological studies, confluent monolayers on Transwell inserts were transferred to HBSS and preincubated with apical CT at 4°C for 30 rain before shifting to fresh HBSS at 37°C. Measurements of short circuit current (Isc) and resistance (R) were performed with 0.33-cm 2 monolayers as previously described (9,28,33). Serosal and mucosal reservoirs were interfaced with calomel and Ag-AgC1 electrodes via 5% agar bridges made with Ringers buffer (114 mM NaCl, 5 mM KCI, 1.65 mM Na2HPO4, 0.3 mM NaH2PO4, 25 mM NaHCO3, 1.1 mM MgSO4, 1.25 mM CaCl2). Measurements of resistance were made using a dual voltage/current clamp device (University of Iowa, Iowa City, Iowa) and 25-~tA current pulses. Isc was calculated using Ohm's law.

Selective Cell Surface Biotinylation
Methods described by Sargiacomo et al. (1989) and Le Bivic et al. (1990) were modified for use in the Tg4 cell system as described (29).

GM1 Binding and ELISA
Fluorescein-labeled CT (CT-fluorescein) (28) was used to define binding of recombinant toxins to GM1 immobilized on plastic micro-titre plates as previously described (30). Dynatec microfluor 96-well black microtitre plates were coated with type III gangliosides (20 Ixg/ml; Sigma Chemical Co.) in 60 mM sodium carbonate, pH 9.9, and incubated at 37°C for 36-48 h as previously described (34). After washing in PBS (140 mM NaCI, 10 mM Na2HPO3/NaH2PO3, pH 7.4) the plates were blocked in PBS containing 5% BSA. CT-fluorescein (3.1 nM in PBS containing 2.5% BSA, pH 7.4) was applied to each well with increasing concentrations (0.05-50 nM) of recombinant wt and mutant toxins or buffer alone, incubated for 30 min at 37°C, washed with PBS, and fluorescence quantified in a fluorescence concentration analyzer (Pandex Fluorescence Plate Reader; Baxter, McGraw Park, IL).
To assay toxin concentrations an EL|SA-based assay was used. Periplasmic extracts containing recombinant toxins were applied to 96-well microtitre plates coated with ganglioside GM1 as previously described (34). After 30 min at 37°C, the plates were washed with PBS, pH 7.4, and the presence of CT or LT bound to GM1 was assayed by routine ELISA using rabbit polyclonal antiserum raised against either CT-A subunit or CT B subunit (27,29). Toxin concentrations were confirmed by SDS-PAGE and Western blot.

Toxin-catalyzed ADP-Ribosylation of Agmatine
NAD:agmatine ADP-ribosyltransferase activity was assayed as described by Tsai (64) with minor modifications. Recombinant wt or K(R)DELmutant toxins (~200 nM final) were incubated at 30°C for 60 rain with 15 mM agmatine, 100 I~M nicotinamide adenine dinucleotide (NAD, containing 0.02 ~Ci [carbonyl-14C]NAD), 1 mM GTP, 50 mM KHzPO4, 4 mM MgCI2, 20 mM dithiothreitol, 0.3 mg/ml ovalbumin, 2 mg/ml cardiolipin, 200 I~M cibachrome blue, and 20 Ixl crude guinea pig brain cytosol (as a source of ADP-ribosylating factor) in a final volume of 100 p.l. Reactions were terminated with the addition of 1 ml 1% SDS in water and applied to AG l-X2 (Bio-Rad Labs., Richmond, CA) 0.25 × 2-cm columns. The columns were washed with 4 ml of distilled water and eluates containing hydrolyzed [carbonyl-14C]nicotinamide were assayed by scintillation counting. Recombinant toxins were first activated by incubation with 30 mM dithiothreitol for 60 min at 30°C. Three point calibration was provided by incubation with dithiothreitol activated purified CT (1 or 0.01 ~M; Calbiochem Novabiochem) or buffer alone. The assay was linear within these toxin concentrations as assessed by formal dose responses.
Guinea pig brain was homogenized (15 strokes in tight fitting Dounce) in 0.34 M sucrose, 0.1 mM MgCI2, 1 mM EDTA, 10 mM Hepes, pH 7.4, containing 1.25 mM PMSF and 0.5 ~l/ml chymostatin. Cytosol was prepared by differential centrifugation and defined as the supernatant of a 100,000 g spin for 60 min.

Endocytosis
Methods described by Orlandi and Fishman were modified for use in the T84 cell system (44). Recombinant toxins (20 nM) were incubated with confluent monolayers of T84 cells grown on plastic 96-well plates at 4°C for 20 min and then shifted to 37°C or kept at 4°C. At the indicated times, monolayers were returned to 4°C and washed. Toxin remaining at the cell surface was measured by immunoreactivity to anti-serum raised against CT B subunit (1:200 dilution) (29), secondary incubation with horseradish peroxidase-labeled anti-IgG, and development with 2, 2'-azino-bis (3-ethylbenzthiazoline)-6-sulfonic acid. Assays were performed in triplicate or quadruplicate and reproduced four times. Two point calibrations for each experiment were provided by incubation at 4°C (positive control) and incubations without CT (negative control, ~30% of maximal signal). Means ---SE are reported.

Preparation of Recombinant CT and LT
Mutations were engineered into the COOH-terminal K(R)DEL sequence of the A subunit of V. cholerae and E. Lencer et al. Targeting of Cholera Toxin by KDEL coli heat-labile enterotoxins. In CT, the COOH-terminal K D E L sequence was replaced by the unrelated sevenamino acid sequence: Leucine-Glutamic acid-Aspartic acid-Glutamic acid-Arginine-Alanine-Serine (Fig. 1). In LT, the COOH-terminal R D E L sequence was altered by a single point mutation replacing leucine with valine ( Fig. 1 B). Both removal of KDEL and substitution of valine for leucine have been shown to inhibit post-ER retrieval of K(R)DEL-containing proteins in other eucaryotic systems (47,48,71). Recombinant wild-type and mutant CT and LT were expressed from plasmid vectors in E. coli XL1-Blue, followed by extraction of periplasmic proteins (containing the recombinant toxins) as described in Materials and Methods. Toxin concentrations were determined by GM~-ELISA against both CT A and pentameric B subunits. Further analysis by SDS-PAGE and Western blot showed that A and B subunits of both mutant and wildtype toxins were present in known concentrations. As assessed by GMI-ELISA, A and pentameric B subunits were present in ~1:1 ratio indicating that all functional recombinant B subunits were fully assembled with A subunits. Further functional analysis of toxin assembly by competitive inhibition with recombinant B subunit confirmed these results (see below).

Mutation in COOH-terminal KDEL Does Not Affect Toxin Binding or ADP-ribosyl-transferase Activity In Vitro
Toxin binding to GMl immobilized on plastic was examined in vitro to determine whether mutation in K(R)DEL may affect indirectly the ability of the toxin B subunits to associate with cell membranes. Fluorescein-labeled CT was utilized to generate binding isotherms for wt and mutant CT and LT. Fig. 2 shows that both the RDEL-(A) and KDEL-mutant (B) bound to GM~ with apparent affini-ties that were nearly identical to that of wt LT and wt CT (Ka -~6 nM). Competition with increasing concentrations of heat denatured CT had no effect on CT-fluorescein binding, indicating that binding was specific.
Enzymatic activities of recombinant toxins (not proteolytically nicked in the exposed loop between the A1 and A2-peptide) were assessed by in vitro ADP-ribosylation of agmatine (64) (Fig. 3). These data show that wt and KDEL-mutant CT (200 nM) displayed nearly identical ADP-ribosyltransferase activities in vitro (1.6 -0.08 vs. 1.8 _+ 0.02 nmol NAD hydrolyzed/h, mean + SD in triplicate, representative of two independent experiments).

Mutation in the COOH-terminal K(R)DEL Sequence Impedes the Time Course of Toxin Action
Initial experiments with T84 cells showed that mutation in the K(R)DEL sequence delayed the onset of toxininduced C1-secretion. At T1/2 (half-maximal secretion: 50 rain for wt CT and 80 rain for wt LT), secretory responses elicited by wild-type toxins were two-to threefold stronger than those elicited by corresponding KDEL-(26 + 3 vs. 8 -+ 2 p~A/cm 2) or RDEL-mutants (25 _+ 0.7 vs. 13 _+ 1 ~A/ cm2; mean _+ SE, n = 6-8). Periplasmic extracts from a control E. coli strain XL1-Blue not harboring toxin clones were electrically silent. Mutation in K(R)DEL affected both the initial lag phase and rate of toxin induced Cl-secretion ( Fig. 4 and Table I). The inhibitory effect on the kinetics of toxin action appeared greater in monolayers exposed to lower toxin concentrations (Fig. 4, C and D) but was otherwise not dose dependent. At T1/2, dose dependencies for both mutant toxins were increased />10fold (Fig. 5). At high toxin concentrations (100 nM), the maximal secretory responses eventually reached were not significantly different (Fig. 4, A   cessing faster than wt CT (Fig. 6). Nearly identical amounts of A and B subunit were immunoprecipitated from T84 cells after 15, 60, and 120 min incubations at 37°C. To exclude the possibility that mutation in K(R)DEL affected toxin action indirectly by affecting assembly of A and B subunits, recombinant LT-B subunits were applied to T84 monolayers in competition with K(R)DEL-mutant or wt toxins (Fig. 7). Recombinant LT B subunit displayed nearly identical IC50's for inhibition of toxin action (IC50 = 10 -4 nM vs. 10 -+ 6 nM, for RDEL-mutant and wt LT, mean + SD, n = 2 in duplicate, upper panel; and IC50 = 11 _ 1 nM vs. 11 _+ 4 nM for KDEL-mutant and wt CT, mean + SD, n = 3 in duplicate, lower panel). These data confirm that mutation in K(R)DEL had no detectable effect on toxin assembly and periplasmic extracts contained little or no free B subunit. Taken together, these data provide evidence that the K(R)DEL motif is not absolutely required for toxin action, but in its absence signal transduction is significantly less efficient. The clear sensitivity displayed by both CT and LT to mutation in K(R)DEL strongly implies that both toxins require functional interaction with endogenous KDEL-receptors within Golgi cisternae or ER for efficient retrograde transport and maximum biologic activity.

Effect of Cell Polarity on Signal Transduction by KDEL-Mutants
When applied to basolateral instead of apical reservoirs, CT can bind to the cell at a site in close proximity to its target enzyme adenylate cyclase. Signal transduction from basolateral cell surfaces, however, also requires endocytosis and subsequent transport of CT through multiple compartments of the central vacuolar network (27,45).
To examine whether signal transduction via basolateral receptors may also require sorting by K(R)DEL, it was necessary to first proteolytically nick (trypsin, 20 lxg/ml) the exposed loop between the A1-and A2-peptides (residues 187-199) (35). The A subunits of the recombinant toxins are not nicked by endogenous proteases in E. coli as they are in V. cholerae (Fig. 1, Western blots). This has no effect on the time course of toxin action when applied to apical cell surfaces in the T84 cell system where both recombinant CT and LT are nicked by a protease(s) at the apical cell surface. Nicking, however, appears to be rate limiting when toxins are applied to basolateral membranes (unpublished data).
Signal transduction by toxin internalization via basolateral endosomes was also sensitive to mutation in the K(R)DEL sorting signal (Fig. 8). KDEL-mutants applied to either apical (A) or basolateral cell surfaces (B) elicited a secretory response more slowly than wild-type CT. Both the initial lag phase and rate of toxin induced C1-secretion were affected (KDEL-mutant vs. wt CT: lag phase, 53 +_ 4 vs. 35 __ 7 min; dlsc/dt, 1.4 -+ 1 vs. 2.5 -+ 1.4 ixA/cm2/ min; peak Isc, 62 _+ 25 vs. 80 _+ 28 ixA/cm 2, mean _+ SD, n = 2 in duplicate). Identical results (qualitatively) were obtained for wt LT and RDEL-mutants (data not shown). Thus, mutation in K(R)DEL affects a physiologic event in the cellular processing of CT that is in common with toxin entry through apical or basolateral endosomes. This common event displays sensitivity to mutation in the K(R)DEL sorting signal and as such represents retrograde traffic of CT and LT through proximal components of the secretory pathway (compartments shared in polarized cells by the apically and basolaterally directed secretory pathway). Thus these data fit nicely with the current hypothesis that translocation of the A subunit (an event essential for toxin action) may occur within Golgi cisternae or ER (50).

Signal Transduction by KDEL-Mutant CT Displays Greater Sensitivity to Temperature
We examined the temperature dependency of toxin induced CI-secretion. We reasoned that if the KDEL sorting signal facilitates a rate-limiting step in toxin action, KDEL-mutants should display greater sensitivity to temperature. Fig. 9 shows  where the ability of the KDEL-mutant to elicit a secretory response was completely inhibited, while C1-secretion induced by wt CT was clearly present. The viability of these monolayers was demonstrated by the brisk secretory response elicited by the administration of 3 nM vasoactive intestinal peptide to basolateral reservoirs at 480 min (marked with * in lower panel). This differential temperature dependency was not dependent on toxin concentration. Identical results were obtained when wt and mutant toxins were applied in fivefold higher concentrations (5 nM). Table II summarizes the results of seven independent experiments. These data provide additional kinetic evidence that specific interactions between CT and endog- enous KDEL-receptors occur in human intestine and play a physiologic role in toxin action.

Effect of KDEL-Mutation on Toxin Traffic: Endocytosis Is Unaffected but Transcytosis of the KDEL-Mutant Occurs More Slowly Than for Wild-type Toxin
To show directly that mutation in K(R)DEL affects toxin trafficking, we examined the time course of endocytosis and transcytosis for both wt and KDEL-mutant toxins. The reduced efficiency in signal transduction by K(R)DELmutants may be attributable to an effect on toxin uptake by endocytosis. The data show, however, that mutation in K(R)DEL had no detectable effect on endocytosis (Fig.  10). Both wt and K(R)DEL-mutant toxins were internalized at nearly identical rates with apparent half-times of ~7.5 min (n = 4, in triplicate or quadruplicate).
The transcytotic movement of wt and KDEL-mutant toxins from apical to basolateral membrane was also examined. In polarized intestinal epithelia, CT must transduce a signal from its apical receptor (GM1) to its basolateral effector (adenylate cyclase). We have recently found that this transcellular signaling may require transcytosis of toxin containing membranes (29). Transcytosis of KDELmutant or wt CT from apical to basolateral membranes was assessed directly by selective cell surface biotinylation. These experiments were carried out at 27°C to enhance the difference in time course between KDEL-mutant and wt CT-induced C1-secretion. Fig, 11 A shows the representative results of one of three independent experiments. The upper panel shows a western blot to demonstrate that each lane contains equivalent amounts of immunoprecipitated CT B subunit. The  CT (B). Representative of two to three independent experiments, mean _+ SD for duplicate monolayers.
lower panel shows the avidin blot of monolayers exposed to KDEL-mutant or wt CT at 27°C for the indicated times. The first and second lanes are positive and negative controis respectively. Wild-type CT (1 nM) was applied apically to T84 monolayers and incubated for 160 min at 4°C, a temperature that completely inhibits vesicular traffic and CT-induced signal transduction (28). The amount of wt and KDEL-mutant toxin transcytosed were quantified (mean _ SE, n = 3) and displayed superimposed at the appropriate points on the time course of CT-induced C1-secretion obtained from monolayers of the same passage and studied in parallel (Fig. 11 B). The  Taken together, these data provide direct evidence that mutation in KDEL affects toxin movement within a basolaterally directed transcytotic pathway and identify Golgi and ER as intermediary compartments through which CT traffics.

Evidence for Retrograde Transport of CT and LT into Golgi Cisternae and ER
The results in this paper show that the presence of a functional K(R)DEL sequence in CT and LT plays a role in facilitating toxin action in human T84 cells and thus implies a direct and specific interaction with endogenous KDELreceptors during signal transduction. In the absence of a functional K(R)DEL sequence at the COOH terminus of the A2-peptide, both mutant CT and LT elicited a secretory response more slowly and with higher apparent activation energies than their wild-type counterparts. The interaction between toxin and KDEL-receptor was specific in that a single and conservative point mutation (replacing leucine with valine in the RDEL sequence of LT) affected toxin action. These results were not explained by indirect effects on toxin assembly, toxin binding, resistance to intracellular degradation, or ADP-ribosyltransferase activity. In contrast, we provide direct evidence that K(R)DEL effected toxin trafficking as assessed by movement through the basolaterally directed transcytotic pathway. The compartment(s) affected represent a common structure in the apical and basolateral endocytic pathways, as mutation in K(R)DEL inhibited signal transduction from both cell surfaces. These data provide strong evidence that CT and LT may require retrograde movement through the secretory pathway to elicit a maximal secretory response. Two lines of evidence from the available literature support this view. First, CT (conjugated to horseradish peroxidase) has been visualized directly within trans-Golgi in neuroblastoma cells (23) and recently, purified CT B subunit (also conjugated to horseradish peroxidase) has been visualized within cis-Golgi in A431 epidermoid cells (56).
These studies show directly that CT can traffic from the cell surface to trans-Golgi and retrograde into Golgi cisternae. Movement into these compartments appears to de- pend on toxin binding (or clustering) ganglioside GM1 , as purified or recombinant CT B subunit will move into these compartments in the absence of sorting signals from the A subunit. These data fit nicely with our current findings that the K(R)DEL sequence is not absolutely required for toxin action, and with our concurrent observations (29) that trafficking of CT through a basolaterally directed transcytotic pathway also depends primarily on toxinbinding (or perhaps clustering) ganglioside GM1. Second, the KDEL-receptor, the product of the mammalian ERD2 gene, has now been clearly localized at steady-state to Golgi cisternae and intermediate compartment with small amounts of receptor visualized in ER and trans-Golgi (16,63). secretory pathway. Furthermore, the preponderance of evidence establishes that KDEL-receptors mediate retrograde transport of other KDEL-containing proteins through Golgi cisternae to ER (42,48,62). Retrieval and recycling to ER occurs from multiple sites in the secretory pathway, including trans-Golgi (8,20,31,37,52). Thus, CT and LT would have multiple opportunities to interact with KDEL receptors as these toxins move retrograde in the secretory pathway via binding (or clustering) ganglioside GM1 (see below). Components of the apical and basolateral endocytic pathways converge in late endosomes (2,12,18,46) and almost certainly in the trans-Golgi, as vesicles exchange constitutively between these compartments (15 and references therein). Our demonstration, however, that the K(R)DEL sorting signal plays a physiologic role in toxin action indicates that both CT and LT move beyond the late endosome into compartments where interactions between toxin and KDEL-receptors may plausibly occur.
Thus, these studies identify trans-Golgi, Golgi cisternae, and possibly ER as intermediary compartments through which CT may physiologically traffic to elicit an efficient secretory response. Since reduction and translocation of the A subunit also represent events that occur in shared (or similar) compartments of both the apical and basolateral endocytic pathways (27), it is tempting to speculate that A subunit translocation occurs in ER or possibly Golgi cisternae as previously hypothesized (50). If so, movement into these compartments would be facilitated by direct interaction between toxin and K(R)DEL-receptors. This prediction fits nicely with our experimental data and provides a cogent and biologically plausible explanation for conservation of the COOH-terminal K(R)DEL sorting signal in these two toxins. However, it remains possible that mutation in K(R)DEL may also affect membrane translocation of the A subunit by a mechanism separate from its effect on toxin trafficking.

Biology of Glycosphingolipids and Targeting of CTILT by Binding or Clustering Ganglioside GM1
Our current studies, together with those discussed above (23,38,56), show that both retrograde traffic into the secretory pathway and transcytosis across polarized cells (29)  golipids such as ganglioside GM1 a r e expressed exclusively on the outer (exo-cytoplasmic) leaflet of cell membranes (reviewed in 57,65,67,68). They contribute to the structure and function of membrane domains throughout the central vacuolar network of eucaryotic cells except perhaps ER where they may be present in extremely low concentrations. Transport between distinct intracellular structures occurs only by vesicular traffic. Thus, ganglioside GM1, which has been shown directly to recycle between the cell surface and trans-Golgi in murine NCTC 2071 and rat glioma C'6 cells (10), represents an ideal target for toxins entering the endocytic and retrograde secretory pathway• Targeting of CT and LT into Golgi and ER (though not efficient) may simply depend on binding (or clustering) GM1 via the pentameric B subunit. This would explain our observations that the K(R)DEL sorting signal is not absolutely essential for toxin action.

Possible Mechanism of CT Action on Polarized Epithelia
Based on our current understanding, we propose the following working model of toxin action on polarized epithelia (Fig. 12). Binding of CT or LT to ganglioside GM1 at the apical cell surface is sufficient for targeting these toxins to all intracellular compartments necessary for signal transduction. In the absence of any other targeting information, however, retrograde movement of CT or LT through the endocytic system to more proximal compartments of the secretory pathway is not efficient. Interaction with endogenous KDEL-receptors shifts the steady state distribution of the CT-GM1 complex towards Golgi cisternae and ER where reduction and translocation of the A subunit may occur. If after translocation the Al-peptide breaks free of the membrane, activation of adenylate cyclase on the basolateral membrane may then occur by diffusion through the cytosol. Available data indicate, however, that the A lpeptide may not dissociate from the membrane after translocation (28,69), and that signal transduction may depend instead on movement of the CT-GM1 complex back out the secretory pathway (via anterograde transport) within basolaterally targeted vesicles (29). Escape of toxin from the KDEL-recycling pathway into anterograde transport vesicles may occur as a result of saturation of KDELreceptors (by incoming toxin) or by sequestration of the K(R)DEL motif after translocation of the A subunit. Fusion of toxin-containing vesicles with the basolateral membrane delivers the Al-peptide to a site where the toxin can now catalyze the ADP-ribosylation of Gsc~. Thus, direct interaction with endogenous KDEL-receptors facilitates toxin entry into Golgi cisternae and ER where reduction and translocation of the A subunit may occur and where the toxin may now transfer from apically recycling to basolaterally targeted vesicles (Fig. 12). Both maximal toxin action and efficient transcytosis then depends on trafficking through these proximal components of the secretory pathway.
Though transcytosis of toxin-containing membranes from apical to basolateral cell surface appears to be required for biologic activity, transcytosis of toxin subunits by itself will not lead to signal transduction (29). Our current and previous studies show that CT must traffic through multiple intracellular compartments to elicit a C1-secretory response.
Here we provide evidence that identifies trans-Golgi, Golgi cisternae, and possibly ER as intermediary compartments through which CT may traffic. Available data indicate that this indirect route across T84 cells may be more complex than that taken by the apically directed polymeric immunoglobulin receptor (41) or by basolateraUy targeted VSG proteins artificially inserted into apical membranes of MDCK cells (51). As such, we introduce the term "indirect transcytosis" to describe this process.
We can now tentatively assign anatomic identities to physiologic compartments previously defined only by functional criteria. For example, brefeldin A inhibits completely CT-induced signal transduction from both the apical and basolateral cell surface. Brefeldin A scrambles protein targeting and diverts the movement of CT away from a compartment necessary for reduction and translocation of the A subunit (27). The brefeldin block occurs early in the lag phase. Based on our current studies, we propose that brefeldin A may inhibit toxin action by inhibiting retrograde transport into trans-Golgi or Golgi cisternae. Either of these sites would be consistent with known effects of brefeldin A on membrane traffic in both polarized and nonpolarized cells (26). Signal transduction from the apical membrane of polarized cells also depends on movement of CT through a separate temperature-sensitive step (27,28). This occurs late in the lag phase after the A subunit has been reduced and presumably translocated. This temperature-sensitive step is not apparent when CT is applied to basolateral cell surfaces, and represents a step(s) uniquely required for signal transduction from apical membranes. We propose that this late lag phase temperature-sensitive step represents sorting (or transfer) of toxin from apically derived to basolaterally targeted membrane structures (i.e., membrane microdomains). This must occur within Golgi cisternae or ER as mutation in K(R)DEL has a clear effect on transcytosis of CT. In contrast, CT internalized from basolateral membranes of the same cells does not display such a dramatic sensitivity to temperature, as signal transduction from the basolateral cell surface does not require movement to the contralateral apical membrane. After arrival within Golgi cisternae or ER, the CT-GM1 complex internalized from the basolateral cell surface can move retrograde into the secretory pathway and back out again to adenylate cyclase on the basolateral membrane by remaining associated with membrane receptors derived from (and presumably targeted to) the basolateral membrane.
In summary, these studies identify Golgi cisternae and ER as intermediary compartments through which CT may traffic to elicit an efficient and maximal physiologic response. The unique lipid and protein structure of these compartments (3) may provide a thermodynamically or kinetically favorable environment for translocation of the A subunit. The COOH-terminal K(R)DEL sorting signal facilitates toxin entry into the secretory pathway, but movement into Golgi cisternae and possibly even ER will occur (though not efficiently) in the absence of the K(R)DELsorting signal after toxin binding (or clustering) ganglioside GM1. Thus CT, as a paradigm for vesicular transport, provides an example where protein targeting (and ultimately the pathogenesis of cholera in humans) depends on the combined effects of protein and lipid structure in small membrane microdomains (58,68).