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Previous Article | Next Article 
Molecular and Cellular Biology, July 2004, p. 5746-5756, Vol. 24, No. 13
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.13.5746-5756.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111
Received 9 October 2003/ Returned for modification 2 December 2003/ Accepted 5 April 2004
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ABSTRACT |
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INTRODUCTION |
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Ral proteins are present in the plasma membrane, but a major fraction of the proteins reside on intracellular vesicles (2, 21, 26). These include components of both the endocytic and exocytic compartments, with both RalA and RalB present at particularly high levels in synaptic vesicles and in platelet granules. It is likely, however, that RalA and RalB have different subcellular distributions because, although both proteins are posttranslationally modified by geranylgeranylation (20), a major difference in their amino acid sequences is in the "variable domain" near their C termini. In other Ras GTPases, this region participates in targeting the proteins to specific membrane compartments.
A family of Ral-specific guanine nucleotide exchange factors that couple Ral activation to a wide variety of upstream signals exist (33). The best-studied example is a set of Ral-specific guanine nucleotide exchange factors that are activated by binding activated Ras. Since Ras is activated by many types of stimuli, Ral proteins also have the potential to participate in mediating the action of a wide variety of extracellular signals. Ral proteins can also be activated by Ras-independent mechanisms, which are not well understood (1, 13, 34). RalA and RalB are identical in regions thought to respond to guanine nucleotide exchange factors, and to date no differences in the responsiveness of RalA and RalB to upstream signals have been reported.
Active RalA has a wide range of effects on cells. It can activate transcription factors, promote cell proliferation, and even contribute to oncogenic transformation (for a review, see reference 9). The mechanism by which Ral influences these processes remains unclear, because the specific downstream target proteins responsible for these effects are not known. However, at least three downstream targets of both Ral proteins that potentially allow Ral proteins to influence vesicle sorting and the actin cytoskeleton have been identified. The first Ral target identified was RalBP1/RLIP (4, 16), which connects Ral proteins with components of the cellular machinery involved in receptor-mediated endocytosis. For example, RalBP1 forms a complex with proteins such as adaptin (17), POB1/Reps1 (18, 44), and epsin and eps15 (22). Although active RalB suppresses receptor-mediated endocytosis (17, 25), how it influences endocytosis through these proteins is not yet clear. Active RalA has since been shown to interact with the actin-binding protein filamin and as such to promote filapodium-like changes in cell morphology (28).
Recently, the exocyst complex has been shown to be another Ral effector (3, 23, 31, 39). The exocyst is a multiprotein complex that contains at least eight different subunits, and active RalA and active RalB have been shown to bind to both the sec5 and exo84 subunits in a GTP-dependent manner (23, 24, 39). The exocyst was first identified in Saccharomyces cerevisiae, where it directs vectorial targeting of secretory vesicles to specific sites on the plasma membrane (for a review, see reference 27). A similar complex also exists in mammalian cells, where it also participates in delivering vesicles to the plasma membrane (14, 19). In polarized epithelial cells, the exocyst participates in the delivery of membrane components destined for the basolateral but not the apical side of the cells (12). In neurons, the exocyst appears to participate in delivering synaptic vesicles (42) and calcium channels, like the N-methyl-d-aspartate glutamate receptor (36) to newly generated synapses, although its abundance in the synaptic endings of adult animals (43) suggests that it may also contribute to the maintenance of synaptic function (31). The exocyst also targets Glut4-containing vesicles to the plasma membrane in response to insulin (15).
How Ral influences secretion through exocyst binding is just beginning to be revealed. Depletion of RalA by interfering RNA expression leads to dissociation of the exocyst complex and a breakdown in polarized membrane delivery (23), suggesting that RalA proteins promote exocyst complex formation. However, for reasons that are not yet clear, expression of a constitutively activated RalB protein caused the same inhibitory phenotype as RalA depletion (23). Interactions of RalA with the exocyst have also been implicated in the formation of filopodia (39). Interestingly, this effect was not due to the expected role of the exocyst in membrane delivery, suggesting that this complex may also influence the actin cytoskeleton associated with filopodium formation.
In this paper we show that active Ral can enhance basolateral delivery of membrane components through the exocyst. However, this is true only for RalA, not for RalB, because the latter has a significantly lower affinity for the exocyst due to a segment of the Ral protein not previously thought to be involved in effector protein binding. Another reason for this difference is that active RalA and RalB localize differently in cells. Finally, the data show that RalA binding to components of the exocyst may not be sufficient to promote secretion, implying that another RalA target protein involved in this process remains to be identified.
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MATERIALS AND METHODS |
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Cell culture. MDCK cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal calf serum (HyClone) supplemented with the necessary antibiotics. To establish inducible cell lines, T23 MDCK cells (a kind gift from J. E. Casanova, University. of Virginia) were cotransfected with a hygromycin resistance plasmid and the pBIG vector (Clontech) with a bidirectional tet-off promoter and lacZ on one side and Ral mutant cDNA inserted on the other side. Cell lines were selected in medium supplemented with 0.25 mg of hygromycin and 0.3 µg of doxycycline per ml. The resulting cell lines were first tested for ß-galactosidase induction, and only cell lines where all cells stained for ß-galactosidase were selected. After that, the cell lines were tested for induction of Ral mutant expression. Cells were maintained in medium supplemented with 0.3 µg of doxycycline per ml.
Synthesis and surface delivery assays. Cells from confluent 100-mm culture dishes were trypsinized, and 25% of the cells were put into 24-mm Transwell filter inserts (0.45-µm pore size; Costar). Cells were grown for 3 days, with the medium being changed daily. Formation of tight junctions was checked by measuring diffusion of fluorescein isothicyanate (FITC)-dextran across the cell monolayer. The cells were starved in DMEM without methionine and cysteine for 1 h and then pulse-labeled by placing 100-µl drop of starvation medium with 2 mCi of 35S-Tran label (ICN) on the sheet of Parafilm in the 37°C water bath and placing filters with cells onto these drops for 15 min; 500 µl of starvation medium with 20 mM HEPES (pH 7.5) was added to upper chambers of filter inserts. Then medium from the upper chambers was removed, and inserts were put into the six-well clusters with 2 ml of regular DMEM/well, and 1 ml of DMEM was added to the upper chambers of the inserts. Cells were then incubated at 37°C for various periods of time.
For polarized delivery assays, filter inserts were washed with ice-cold Dulbecco's phosphate-buffered saline (PBS), and apical or basal surfaces were biotinylated on ice for 30 min with 1 mg of Sulfo-SS-NHS-biotin (Pierce) per ml in Dulbecco's PBS. Biotinylation was stopped by washing filters twice with ice-cold Tris-buffered Dulbecco's PBS, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, and E-cadherin or gp135 was immunoprecipitated. Beads with antibody-antigen complex were then boiled with 20 µl of 3% sodium dodecyl sulfate (SDS), diluted with 1 ml of reprecipitation buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mmM EDTA, 1% Triton X-100), supernatant was reprecipitated with avidin beads, and precipitated proteins were then run in SDS gels and analyzed by phosphoimager analysis.
For assays of E-cadherin processing, after chase filter inserts were washed with ice-cold Dulbecco's PBS, cells were lysed in RIPA buffer and cadherin was immunoprecipitated with anticadherin polyclonal antibody. Immunoprecipitates were run on SDS-protein gels, dried, and subjected to phosphoimager analysis.
Immunofluorescence. Cells were induced or uninduced for expression of Ral mutants and seeded onto polylysine-treated 10-mm coverslips. Cells were washed with cold PBS, fixed with 4% paraformaldehyde in PBS, permeabilized with 0.075% Saponin in PBS for 15 min, and blocked with 1% bovine serum albumin in PBS with 0.075% saponin. Cells were then incubated with primary antibody in PBS with 1% bovine serum albumin and 0.075% saponin for 2 h at room temperature, washed with PBS, and incubated with secondary antibody in PBS with 1% bovine serum albumin and 0.075% saponin for 1 h at room temperature, washed three times with PBS, and mounted in PBS with 50% glycerol and 0.2% n-propylgallate.
For colocalization of Sec6 and RalA, cells were fixed with either paraformaldehyde or methanol, permeabilized as described above, and incubated sequentially with intermittent washes with the following antibodies, all in 1% bovine serum albumin-0.075% saponin in PBS: mouse anti-Sec6 antibody, donkey anti-mouse Fab-Cy2, excess unlabeled donkey anti-mouse Fab, mouse anti-RalA antibody, and donkey anti-mouse Fab-Cy3. Cells were then washed and mounted as described above. Images were acquired with an Axioplan (Carl Zeiss) microscope and 40x objective equipped with a digital camera and processed with Adobe Photoshop 6.0.1 for Macintosh.
Loading of recycling endosomes with transferrin. RalA72L T23 MDCK cells were induced for RalA72L expression, infected with adenovirus expressing human transferrin receptor, and sparsely plated onto polylysine-treated 10-mm coverslips. Coverslips with cells were incubated in serum-free DMEM for 40 min, inverted onto 100-µl drops of DMEM with 0.1 mg of Texas Red-labeled human transferrin (Molecular Probes) per ml on the Parafilm sheets on ice, and incubated on ice for 20 min to allow transferrin binding. Coverslips were then transferred to 37°C DMEM with 5% fetal calf serum and chased for 20 min. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.075% saponin, and processed for RalA staining as described above.
Images were acquired with a Leica TCS SP2 confocal microscope and 63x objective and processed with Adobe Photoshop 6.0.1 for Macintosh.
Immunoprecipitation. For coimmunoprecipitation experiments, 293T cells were seeded at 50% confluency on a 100-mm dish and transfected with N-terminally Myc-tagged cDNAs of RalA or RalB mutants with Lipofectamine 2000 (Invitrogen), and 48 h later the cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (20 mM Tris [pH 7.5], 50 mM NaCl, 1 mM MgCl2, 0.5% Triton X-100, freshly supplemented with protease inhibitors). RalA and RalB mutants were immunoprecipitated from 900 ul of lysate with 2 µg of anti-Myc 9E10 monoclonal antibody (Tufts University/New England Medical Center facility) prebound to 15 µl of protein A-Sepharose beads. The beads were washed three times with lysis buffer, boiled with SDS sample buffer, run in SDS gels, transferred to nitrocellulose membranes, and probed with the appropriate antibodies.
For high-salt wash beads with immunoprecipitated Ral mutants were washed twice with 1 ml of high-salt buffer (lysis buffer supplemented with 900 mM NaCl) and then twice with lysis buffer and either boiled with SDS sample buffer or incubated with lysate from untransfected 293T cells for 2 h at 4°C and then washed three times with lysis buffer, boiled with SDS sample buffer, run in SDS gels, and processed for Western blotting with the appropriate antibodies.
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RESULTS |
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Transient induction of active RalA 72L expression in MDCK cells resulted in an enhanced rate (
2-fold) of delivery of E-cadherin to the basolateral membrane compared to its rate of delivery in uninduced control cells (Fig. 2A). The rate of synthesis of E-cadherin was not changed significantly during this time period in induced cells (data not shown). E-cadherin is initially synthesized as a 130-kDa precursor that is cleaved in the trans-Golgi compartment to form a mature 120-kDa protein (38). The kinetics of this processing were unchanged upon expression of RalA 72L (Fig. 2B), indicating that RalA enhances delivery of vesicles containing E-cadherin between the trans-Golgi and the basolateral plasma membrane. The effect of RalA was not limited to E-cadherin, since investigation of the entire population of newly synthesized basolateral proteins labeled with biotin and then isolated by avidin binding revealed enhanced delivery of many proteins (Fig. 2C). Importantly, the rate of delivery of gp135 (Fig. 2D) and other newly synthesized biotinylated proteins to the apical surface (Fig. 2C) was not significantly different after induction of RalA72L expression.
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To support the idea that RalA interaction with the exocyst is involved in its ability to influence cellular secretion, effector domain mutants of Ral, previously identified on the basis of their loss of binding affinity for specific Ral effector proteins, were investigated. In particular, inducible cell lines expressing RalA72L49E, which fails to bind to the Sec5 and Exo84 subunits of the exocyst (23, 24), or RalA72L49N, which retains the ability to bind to both exocyst components (23, 24, 31), were generated and tested in secretion assays as described above (Fig. 3A). Expression of RalA72L49E failed to promote enhanced delivery of E-cadherin to the basolateral surface of MDCK cells (Fig. 3B). Thus, as expected, exocyst binding to active RalA is needed for the GTPase to enhance secretion. Surprisingly, expression of RalA72L49N also failed to enhance basolateral membrane delivery (Fig. 3B), despite the fact that this mutant can still bind to the exocyst. These findings suggest that exocyst binding is necessary but not sufficient for RalA to enhance secretion.
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5-fold). In fact, by analyzing silver-stained gels of immunoprecipitates, all of the exocyst components that we detected previously bound to active RalA (31), including Exo84, were present at much lower levels on immunoprecipitates of active RalB than active RalA (data not shown). Although active RalB bound less well to the exocyst complex, it still displayed activity-dependent binding (compare RalB72L to RalB28N). Figure 4C shows that all Ral proteins were expressed at similar levels. Moreover, both RalA72L and RalB72 were present almost exclusively in the GTP-bound state in cells (data not shown).
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By aligning the amino acid sequences of RalA and RalB, it becomes clear that the major distinctions between the two proteins are in the segments just distal to the switch 2 effector-binding domain (amino acids 91 to 153) and the variable domain near the C terminus, outside the core GTPase domain (Fig. 5A). The two amino acid differences in the N-terminal extension are not involved in binding differences between RalA and RalB, because their deletion had no effect on exocyst binding (data not shown). To determine which of the other regions are involved, the variable domains of RalA and RalB were switched by making chimeric Ral/RalB proteins at amino acid 176. The chimeric proteins were tested in binding assays as described above (Fig. 5B). The RalA/RalB(variable domain) chimera retained high-affinity binding to Sec5 and Sec8 in cells. The RalB/RalA(variable domain) chimera retained lower binding ability than RalA, although its binding activity did rise. These results indicate that the region between the switch 2 region of the "effector domain" and the variable domain (amino acids 91 to 153) plays a major role in the differences in the exocyst binding potential of RalA and RalB. In addition, the variable domain may also contribute, possibly by altering the localization of the proteins (see below).
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RalB antibody staining of RalB72L-expressing cells also yielded intense staining of the plasma membrane between cells (Fig. 8G). Importantly, however, no accumulation of active RalB was observed in perinuclear regions (Fig. 8G).
Since this perinuclear staining was reminiscent of recycling endosomes, this vesicle population was stained by exposing MDCK cells (infected with human transferrin receptor-encoding virus) with Texas Red-conjugated transferrin and allowing uptake for 20 min (37). A cross-section of cells viewed by confocal microscopy displayed a very similar staining pattern for transferrin and activated RalA (Fig. 9A to C). In contrast, significantly less colocalization of active RalA and the trans-Golgi marker TGN-38 was observed (Fig. 9D to F), suggesting that active RalA but not active RalB accumulates predominantly in recycling endosomes.
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Analysis of the chimeric RalA/RalB proteins was also revealing (Fig. 9I and J). Both chimeras also failed to accumulate in recycling endosomes. Since these mutants were defective in enhancing secretion, these results reinforce the significance of the recycling endosome localization of active RalA for its effects on basolateral delivery of membrane proteins. The failure of the RalA72L/RalB(variable domain) chimera to localize correctly also demonstrates that, as expected, the variable region also participates in localizing active RalA to a site in the cell where it can promote basolateral membrane delivery.
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DISCUSSION |
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In particular, active RalA binds to the exocyst complex much more efficiently than active RalB does. Consistent with this difference, only active RalA, not active RalB, promotes vesicle trafficking expected of a regulator of exocyst function, i.e., delivery of membrane proteins to the basolateral surface of cells. The finding of a difference in intrinsic target protein binding potential between active RalA and active RalB is surprising because they have identical effector domains that in other GTPases determine binding to target proteins. Recent structural analysis shows that a major binding interaction between RalA and Sec5 of the exocyst occurs at residues 38 to 47 of the switch 1 region of the GTPase (11). Other GTPases also interact with targets through their switch 2 regions, although this could not be assessed because that region of RalA is not resolved well in the crystal structure. However, in the present work we find that the binding difference between RalA and RalB was encoded in RalA-specific amino acids 91 to 153, whereas no direct interactions between Sec5 and RalA are present in the structural study.
Interestingly, some of the amino acids that distinguish RalA and RalB in this region are found in helix 3. Our previous study on the generation of the specificity of interactions between exchange factors and the closely related H-Ras and R-Ras GTPases suggested that the switch 2 regions in the effector domains of those proteins are influenced by differences in amino acids in their helix 3 regions (41). Thus, it is possible that differences in the intramolecular interactions between helix 3 and the effector domains of RalA and RalB cause the observed differences in their ability to bind to exocyst components.
Alternatively, amino acids between 91 to 153 may bind to an additional site on Sec5 or to another exocyst component such as Exo84 to generate high-affinity binding, since the structure experiments used only a small part of the Sec5 protein, while we studied binding to the exocyst complex in cells, not to individual components. Since the structure of amino acids 91 to 153 of RalA is not affected by nucleotide binding, this region is not likely involved in nucleotide-dependent binding of the exocyst, but rather in promoting high-affinity binding required for biological activity in cells. This model is consistent with the fact that although RalB binds with relatively low affinity to the exocyst, it still does so in a GTP-dependent manner. Support for this model comes from structural studies on the interaction of Rab3A with its effector rabphilin (30). In this report, additional binding sites for rabphilin outside the effector domain that are proposed to contribute to Rab family member binding specificity. Interestingly, one of these sites is in a region analogous to the one that we have implicated in RalA-exocyst binding.
Immunofluorescence experiments in this study show that active RalA72L and active RalB72L distribute differently in cells. Both active RalA and active RalB are enriched in the plasma membrane between MDCK cells, along with the components of the exocyst complex. However, active RalA also accumulates in a perinuclear compartment that appears to be the recycling endosome, based on its complete colocalization with internalized transferrin and less consistent colocalization with the trans-Golgi marker TGN 38. In contrast, active RalB does not. Although early studies implied that exocyst proteins function at the plasma membrane (12, 45), new studies implicated it in recycling endosomes (10, 32), and we find significant overlap of active RalA and exocyst component Sec6 in this perinuclear region.
The importance of this localization to RalA function is supported by the fact that all Ral proteins studied that do not promote basolateral secretion also do not accumulate in recycling endosomes. These inactive Ral proteins include active RalB72L, effector-domain mutants of RalA72L, and chimeras of RalA72L and RalB72L. The fact that active RalA, which enhances the membrane delivery of newly synthesized proteins, localizes to recycling endosomes, a compartment also known to be involved in endocytosis, highlights the idea that RalA and its targets may function at the junction of exocytosis and endocytosis regulation. Adding support to this notion is our recent finding that another recycling endosome protein, Rab11-FIP2, is part of a signaling complex associated with RalBP1, a Ral effector implicated in endocytosis (8).
Furthermore, we find that while the interaction of Ral with the exocyst is necessary for enhanced secretion, it may not be sufficient because an effector domain mutant of RalA (RalA72L49N) that still interacts with the Sec5 and Exo84 in cells does not enhance the rate of basolateral membrane delivery of vesicle components. This mutant also lacks the ability to accumulate in recycling endosomes. Since this effector mutant does not bind to RalBP1, RalBP1 or a novel Ral effector with similar Ral-binding properties may function with the exocyst to promote secretion.
The fact that the effector-binding domain contributes to the subcellular localization of a GTPase is consistent with that GTPase's functioning in vesicle sorting, since an effector protein may be used to target a GTPase-containing vesicle to a specific site in the cell. However, for Ral proteins, the variable domain near the C terminus of the Ral proteins also plays a key role in the specific localization of active RalA, since this region, not the effector domain, is different between RalA and RalB. In fact, we find that the chimeric protein containing RalA with the variable region of RalB does not colocalize to the recycling endosome even though it maintains high-affinity binding potential for the exocyst. Thus, the variable region and the effector domain likely work in concert to place the active form of the protein in the correct location for active RalA to promote basolateral membrane delivery.
The data from this study also suggests that an additional Ral effector protein, besides the two exocyst components already identified, is needed for correct localization of active RalA and for secretion regulation. Since all of these effectors require interaction with the same effector-binding domain on Ral, it is likely that they cannot bind simultaneously. Thus, proper localization of active RalA may require sequential binding of multiple effectors, possibly one early in the secretory process in recycling endosomes and one at the plasma membrane.
The levels of constitutively activated Ral proteins used in these experiments are comparable to those of their endogenous GTPase counterparts. Thus, they represent the most active Ral protein that could ever exist in an MDCK cell. Nevertheless, this level of active RalB does not enhance basolateral membrane delivery of E-cadherin or other biotinylatable membrane proteins, indicating that RalB is not a regulator of exocyst function, at least for basolateral membrane protein delivery in MDCK cells. A previous study showed that when active RalB is expressed at levels many times higher than endogenous RalB in MDCK cells it still fails to enhance basolateral delivery of membrane proteins (23). In fact, not only do these high levels of active RalB fail to enhance polarized secretion, they actually disrupt polarized secretion, since basolateral membrane proteins such as epidermal growth factor receptor are found on the apical side of the membrane. This disruption, which we do not observe when activated RalB is expressed at levels comparable to endogenous RalB (Fig. 6B), is also observed when endogenous Ral activity is blocked in cells (23), showing that high levels of active RalB actually inhibit exocyst-related function.
Our studies suggest that high levels of activated RalB expression overcomes the relatively weak binding of RalB to the exocyst in cells. As such, it targets a significant enough fraction of the exocyst to a nonfunctional site in the cell to disrupt exocyst function. This explanation is consistent with our finding that placing the RalB variable domain on active RalA blocks its activity in cells despite that fact that the protein maintains high affinity for the exocyst.
This model can also explain why Moskalenko et al. (23) found that a D49N mutation, which blocks RalBP1 but not exocyst binding, has no effect on the ability of active RalB to disrupt polarized secretion, while we find that the same mutation suppresses the ability of active RalA to enhance polarized secretion. This difference would be expected if active RalB does, in fact, prevent polarized secretion by removing the exocyst from its active site in cells, since such a "dominant negative" effect would be expected to require the removal of only one critical effector from its natural location. In contrast, the present study suggests that a positive effect of RalA on secretion may require interaction of RalA with more than one effector protein.
The fact that active RalA enhances polarized secretion of many basolateral proteins together with the finding in a previous study that suppression of RalA protein expression inhibits polarized secretion of multiple basolateral proteins (23) firmly implicates RalA as a positive regulator of a key cellular process that contributes to the maintenance of polarity in epithelial cells. Additional studies, however, will be needed to explain how RalA enhances basolateral membrane delivery and secretion.
Finally, if RalB does not regulate secretion, what is its function in cells? A body of evidence has implicated Ral proteins and their target RalBP1 in an early step in receptor-mediated endocytosis (17, 22, 25). Interestingly, all of theses experiments used activated RalB, not RalA, to show an effect on receptor uptake. Thus, it is interesting to speculate that although RalA and RalB have identical effector-binding domains, differences in their ability to bind specific effector proteins and differences in their subcellular localization allow them to carry out unique functions. A recent study has shown, with short interfering RNA-induced depletion of individual Ral isoforms, that RalA is required for the growth of tumor cells in suspension, while RalB is required for tumor cell survival (7). Whether the differences in the biochemical and biological activities of RalA and RalB identified here are related to these intriguing differences in how the two isoforms affect tumor cell growth remains to be determined.
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ACKNOWLEDGMENTS |
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We thank, J. Casanova for tetracycline-inducible MDCK cells, G. Banting, Y. Ohta, W. J. Nelson, and G. K. Ojakian for kindly providing antibodies, and I. Mellman for providing adenovirus expressing the human transferrin receptor.
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FOOTNOTES |
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