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Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B–dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B–dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

We have used temperature and nocodazole blocks in an in vivo basolateral to apical transcytosis assay to dissociate the early transcytotic steps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([Fab]2 fragment of IgG against the receptor's ectodomain) at 17 degrees C but do not deliver it to the apical PM. Instead, the ligand accumulates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule- requiring translocation step. We have characterized this compartment and show that it is distinct from basolateral transferrin recycling endosomes, basolateral early endosomes or late endosomes or lysosomes. The apical transcytotic compartment colocalizes with the compartment containing apically recycling membrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid phase marker (BSA). This compartment is an intermediate station of the overall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17 degrees C are subsequently incubated at 37 degrees C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of trimeric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gs alpha, to cells preloaded with transcytotic ligand at 17 degrees C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and function of the apical endosome are not affected since neither BFA nor cholera toxin inhibit the recycling of apically internalized membrane markers (ricin and pIgA-R ligand) from the same compartment. The data suggest that transcytosis connects the "membrane-sorting" sub-domain of the basolateral endosome with a homologous sub-domain of the apical endosome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, distinct from that used for recycling of apically internalized proteins (ricin or pIgA-R).

When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (≤2.5 min at 37°C) and are then rapidly segregated from one another. The dextran remains in the large supranuclear EEA1-positive early endosomes while recycling polymeric immunoglobulin receptor–bound immunoglobulin A is delivered to a Rab11-positive subapical recycling compartment. This latter step requires an intact microtubule cytoskeleton. Receptor-bound transferrin, a marker of the basolateral recycling pathway, has limited access to the fluid-rich apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome.

Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.

Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the μ1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.

The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Δ, sec5Δ, and exo70Δ strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395–4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Recent studies have identified caveolin-1, a protein best known for its functions in caveolae, in apical endocytic recycling compartments in polarized epithelial cells. However, very little is known about the regulation of caveolin-1 in the endocytic recycling pathway. To address this question, in the current study we compared the relationship between compartments enriched in sub-apical caveolin-1 and Rab11a, a well-defined marker of apical recycling endosomes, using polarized MDCK cells as a model. We show that caveolin-1-containing vesicles define a compartment that partially overlaps with Rab11a, and that the distribution of subapical caveolin-1 and Rab11a show a similar dependence on microtubule disruption. Mutants of the Rab11a effector, Rab11-FIP2 also altered the localization of caveolin-1. These findings indicate that caveolin-1 is coordinately regulated with Rab11a within the apical recycling system of polarized epithelial cells, suggesting that the two proteins are components of the same pathway.

In polarized Madin-Darby canine kidney (MDCK) cells, the transferrin receptor (TR) is selectively delivered to the basolateral surface, where it internalizes transferrin via clathrin-coated pits and recycles back to the basolateral border. Mutant tailless receptors are sorted randomly in both the biosynthetic and endocytic pathways, indicating that the basolateral sorting of TR is dependent upon a signal located within the 61–amino acid cytoplasmic domain. To identify the basolateral sorting signal of TR, we have analyzed a series of mutant human TR expressed in MDCK cells. We find that residues 19–41 are sufficient for basolateral sorting from both the biosynthetic and endocytic pathways and that this is the only region of the TR cytoplasmic tail containing basolateral sorting information. The basolateral sorting signal is distinct from the YTRF internalization signal contained within this region and is not tyrosine based. Detailed functional analyses of the mutant TR indicate that residues 29–35 are the most important for basolateral sorting from the biosynthetic pathway. The structural requirements for basolateral sorting of internalized receptors from the endocytic pathway are not identical. The most striking difference is that alteration of G31DNS34 to YTRF impairs basolateral sorting of newly synthesized receptors from the biosynthetic pathway but not internalized receptors from the endocytic pathway. Also, mutations have been identified that selectively impair basolateral sorting of internalized TRs from the endocytic pathway without affecting basolateral sorting of newly synthesized receptors. These results imply that there are subtle differences in the recognition of the TR basolateral sorting signal by separate sorting machinery located within the biosynthetic and endocytic pathways.

Summary

The transcytotic pathway allows for the bidirectional transport of endocytosed solutes, lipids, and proteins between the two membrane domains of polarized epithelial cells while maintaining the functional integrity of the epithelial tissue. A method is described to measure basolateral-to-apical transcytosis of immunoglobulin A (IgA) in polarized Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor (pIgR). The cells are grown on porous Transwell filter supports, and radiolabeled 125I-immunoglobulin A (IgA) is internalized from the basolateral pole of MDCK cells. During a subsequent 2-h chase, the amount of 125I-IgA that is recycled, degraded, or transcytosed is quantified. This assay can be adapted to follow the postendocytic fate of other 125I-labeled ligands and proteins.

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.

Protein delivery across polarized epithelia is controlled by receptor-mediated transcytosis. Many studies have examined basolateral-to-apical trafficking of polymeric IgA (pIgA) by the polymeric immunoglobulin receptor (pIgR). Less is known about apical-to-basolateral transcytosis, the direction the neonatal Fc receptor (FcRn) transports maternal IgGs across intestinal epithelia. To compare apical-to-basolateral and basolateral-to-apical transcytosis, we co-expressed FcRn and pIgR in Madin-Darby canine kidney (MDCK) cells and used pulse-chase experiments with confocal microscopy to examine transport of apically applied IgG Fcγ and basolaterally applied pIgA. Fcγ and pIgA trafficking routes were initially separate but intermixed at later chase times. Fcγ was first localized near the apical surface, but became more equally distributed across the cell, consistent with concomitant transcytosis and recycling. By contrast, pIgA transport was strongly unidirectional: pIgA shifted from near the basolateral surface to an apical location with increasing time. Some Fcγ and pIgA fluorescence colocalized in early (EEA1-positive), recycling (Rab11a-positive), and transferrin (Tf)-positive common/basolateral recycling endosomes. Fcγ became more enriched in Tf-positive endosomes with time, whereas pIgA was sorted from these compartments. Live-cell imaging revealed that vesicles containing Fcγ or pIgA shared similar mobility characteristics and were equivalently affected by depolymerizing microtubules, indicating that both trafficking routes depended to roughly the same extent on intact microtubules.

The exocyst -- an octameric protein complex mediating vesicle tethering at the plasma membrane for exocytosis -- is a downstream effector of the Rab proteins Rab8 and Rab11, which are key regulators of membrane trafficking from the trans-Golgi network and recycling endosome to the plasma membrane. Rab11 and Rab8 coordinate their actions via Rabin8, the guanine nucleotide exchange factor of Rab8. A cascade of protein-protein interactions involving the Rabs and the exocyst complex couples the generation of secretory vesicles at donor compartments to their docking and fusion at the plasma membrane. Here, we discuss recent work implicating Rab proteins and the exocyst in primary ciliogenesis and epithelial lumenogenesis. In addition, we discuss early work in the budding yeast Saccharomyces cerevisiae, which provided initial insight into the molecular mechanisms of polarized exocytosis.

The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.

Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD μ1A or μ1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B–containing membranes that were both distinct from AP-1A–positive TGN elements and more closely apposed to transferrin receptor–positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.

Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.