RAB-10 is required for endocytic recycling in the Caenorhabditis elegans intestine

The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. One of the most enigmatic aspects of this process is endocytic recycling, the return of macromolecules (often receptors) and fluid from endosomes to the plasma membrane. We have previously shown that the EH-domain protein RME-1 is a critical regulator of endocytic recycling in worms and mammals. Here we identify the RAB-10 protein as a key regulator of endocytic recycling upstream of RME-1 in polarized epithelial cells of the Caenorhabditis elegans intestine. rab-10 null mutant intestinal cells accumulate abnormally abundant RAB-5-positive early endosomes, some of which are enlarged by more than 10-fold. Conversely most RME-1-positive recycling endosomes are lost in rab-10 mutants. The abnormal early endosomes in rab-10 mutants accumulate basolaterally recycling transmembrane cargo molecules and basolaterally recycling fluid, consistent with a block in basolateral transport. These results indicate a role for RAB-10 in basolateral recycling upstream of RME-1. We found that a functional GFP-RAB-10 reporter protein is localized to endosomes and Golgi in wild-type intestinal cells consistent with a direct role for RAB-10 in this transport pathway.     

RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization

Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth.     

Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions.     

RAB-6.1 and RAB-6.2 Promote Retrograde Transport in C. elegans

Retrograde transport is a critical mechanism for recycling certain membrane cargo. Following endocytosis from the plasma membrane, retrograde cargo is moved from early endosomes to Golgi followed by transport (recycling) back to the plasma membrane. The complete molecular and cellular mechanisms of retrograde transport remain unclear. The small GTPase RAB-6.2 mediates the retrograde recycling of the AMPA-type glutamate receptor (AMPAR) subunit GLR-1 in C. elegans neurons. Here we show that RAB-6.2 and a close paralog, RAB-6.1, together regulate retrograde transport in both neurons and non-neuronal tissue. Mutants for rab-6.1 or rab-6.2 fail to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. Loss of both rab-6.1 and rab-6.2 results in an additive effect on GLR-1 retrograde recycling, indicating that these two C. elegans Rab6 isoforms have overlapping functions. MIG-14 (Wntless) protein, which undergoes retrograde recycling, undergoes a similar degradation in intestinal epithelia in both rab-6.1 and rab-6.2 mutants, suggesting a broader role for these proteins in retrograde transport. Surprisingly, MIG-14 is localized to separate, spatially segregated endosomal compartments in rab-6.1 mutants compared to rab-6.2 mutants. Our results indicate that RAB-6.1 and RAB-6.2 have partially redundant functions in overall retrograde transport, but also have their own unique cellular- and subcellular functions.     

Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit

Here we identify a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains Ras-GAP (GTPase-activating protein)-like and Vps9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to Caenorhabditis elegans RAB-5 in the GDP-bound conformation, and rme-6 mutants have phenotypes that indicate low RAB-5 activity. However, unlike other Rab5-associated proteins, a rescuing green fluorescent protein (GFP)-RME-6 fusion protein primarily localizes to clathrin-coated pits, physically interacts with alpha-adaptin, a clathrin adaptor protein, and requires clathrin to achieve its cortical localization. In rme-6 mutants, transport from the plasma membrane to endosomes is defective, and small 110-nm endocytic vesicles accumulate just below the plasma membrane. These results suggest a mechanism for the activation of Rab5 in clathrin-coated pits or clathrin-coated vesicles that is essential for the delivery of endocytic cargo to early endosomes.     

Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells

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.     

Rab8 regulates basolateral secretory, but not recycling, traffic at the recycling endosome

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 mu1-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.     

CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.     

UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in Caenorhabditis elegans

Small guanosine triphosphatases of the Rab family regulate intracellular vesicular trafficking. Rab2 is highly expressed in the nervous system, yet its function in neurons is unknown. In Caenorhabditis elegans, unc-108/rab-2 mutants have been isolated based on their locomotory defects. We show that the locomotion defects of rab-2 mutants are not caused by defects in synaptic vesicle release but by defects in dense core vesicle (DCV) signaling. DCVs in rab-2 mutants are often enlarged and heterogeneous in size; however, their number and distribution are not affected. This implicates Rab2 in the biogenesis of DCVs at the Golgi complex. We demonstrate that Rab2 is required to prevent DCV cargo from inappropriately entering late endosomal compartments during DCV maturation. Finally, we show that RIC-19, the C. elegans orthologue of the human diabetes autoantigen ICA69, is also involved in DCV maturation and is recruited to Golgi membranes by activated RAB-2. Thus, we propose that RAB-2 and its effector RIC-19 are required for neuronal DCV maturation.     

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans

Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function.     

The recycling endosome protein RAB-10 promotes autophagic flux and localization of the transmembrane protein ATG-9

Macroautophagy/autophagy involves the formation of an autophagosome, a double-membrane vesicle that delivers sequestered cytoplasmic cargo to lysosomes for degradation and recycling. Closely related, endocytosis mediates the sorting and transport of cargo throughout the cell, and both processes are important for cellular homeostasis. However, how endocytic proteins functionally intersect with autophagy is not clear. Mutations in the DAF-2/insulin-like IGF-1 (INSR) receptor at the permissive temperature result in a small increase in GFP::LGG-1 foci, i.e. autophagosomes, but a large increase at the nonpermissive temperature, allowing us to control the level of autophagy. In a RNAi screen for endocytic genes that alter the expression of GFP::LGG-1 in daf-2 mutants, we identified RAB-10, a small GTPase that regulates basolateral endocytosis. Loss of rab-10 in daf-2 mutants results in more GFP::LGG-1-positive foci at the permissive, but less GFP::LGG-1 or SQST-1::GFP foci at the nonpermissive temperature. As previously reported, loss of rab-10 alone resulted in an increase of GFP:LGG-1 foci. Exposure of rab-10 mutant animals to chloroquine, a known inhibitor of autophagic flux, failed to increase the number of GFP::LGG-1 foci. Moreover, colocalization between LMP-1::tagRFP and GFP::LGG-1 (the lysosome and autophagosome reporters) was decreased in daf-2; rab-10 dauers at the nonpermissive temperature. Intriguingly, RAB-10 was required to maintain the normal size of GFP::ATG-9-positive structures in daf-2 mutants at both the permissive and nonpermissive temperature. Finally, we found that RAB-10 GTPase cycling was required to control the size of GFP::ATG-9 foci. Collectively, our data support a model where rab-10 controls autophagic flux by regulating autophagosome formation and maturation.     

An endocytic pathway as a target of tubby for regulation of fat storage

The tubby loci provide a unique opportunity to study adult-onset obesity. Mutation in either mammalian tubby or its homologue in Caenorhabditis elegans, tub-1, results in increased fat storage. Previously, we have shown that TUB-1 interacts with a new Rab GTPase-activating protein, RBG-3, for the regulation of fat storage. To understand further the molecular mechanism of TUB-1, we identified the Rab GTPase downstream of RBG-3. We found that RBG-3 preferentially stimulates the intrinsic GTPase activity of RAB-7 in both human and C. elegans. Importantly, either mutation or RNA interference knockdown in rab-7 reduces stored fat in wild type and tub-1 mutants. In addition, the small GTPase rab-5 and genes that regulate Rab membrane localization and nucleotide recycling are required for the regulation of fat storage, thereby defining a role for endocytic recycling in this process. We propose that TUB-1 controls receptor or sensory molecule degradation in neurons by regulating a RAB-7-mediated endocytic pathway.     

Functional Separation of Endosomal Fusion Factors and the Class C Core Vacuole/Endosome Tethering (CORVET) Complex in Endosome Biogenesis

Transport along the endolysosomal system requires multiple fusion events at early and late endosomes. Deletion of several endosomal fusion factors, including the Vac1 tether and the Class C core vacuole/endosome tethering (CORVET) complex-specific subunits Vps3 and Vps8, results in a class D vps phenotype. As these mutants have an apparently similar defect in endosomal transport, we asked whether CORVET and Vac1 could still act in distinct tethering reactions. Our data reveal that CORVET mutants can be rescued by Vac1 overexpression in the endocytic pathway but not in CPY or Cps1 sorting to the vacuole. Moreover, when we compared the ultrastructure, CORVET mutants were most similar to deletions of the Rab Vps21 and its guanine nucleotide exchange factor Vps9 and different from vac1 deletion, indicating separate functions. Likewise, CORVET still localized to endosomes even in the absence of Vac1, whereas Vac1 localization became diffuse in CORVET mutants. Importantly, CORVET localization requires the Rab5 homologs Vps21 and Ypt52, whereas Vac1 localization is strictly Vps21-dependent. In this context, we also uncover that Muk1 can compensate for loss of Vps9 in CORVET localization, indicating that two Rab5 guanine nucleotide exchange factors operate in the endocytic pathway. Overall, our study reveals a unique role of CORVET in the sorting of biosynthetic cargo to the vacuole/lysosome.     

Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo

We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.     

Rab17 and Rab18, Small GTPases with Specificity for Polarized Epithelial Cells: Genetic Mapping in the Mouse

The Rab subfamily of small GTPases plays an important role in the regulation of membrane traffic in eukaryotic cells. While most Rab proteins are equally expressed in polarized and nonpolarized cells, Rab17 and Rab18 show epithelial cell specificity. Here we report the genetic mapping of Rab17 and Rab18 on mouse chromosomes 1 and 18, respectively. We also discuss some implications ofRab17andRab18mapping, including their candidacy for the mouse mutationsln(leaden),Tw(twirler), andax(ataxia).     

The SM protein VPS-45 is required for RAB-5-dependent endocytic transport in Caenorhabditis elegans

Rab5, a small guanosine triphosphatase, is known to regulate the tethering and docking reaction leading to SNARE (soluble NSF attachment protein receptors)-mediated fusion between endosomes. However, it is uncertain how the signal of the activated Rab5 protein is transduced by its downstream effectors during endosome fusion. Here, we show that the Sec1/Munc18 gene vps-45 is essential for not only viability and development but also receptor-mediated and fluid-phase endocytosis pathways in Caenorhabditis elegans. We found that VPS-45 interacts with a Rab5 effector, Rabenosyn-5 (RABS-5), and the mutants of both vps-45 and rabs-5 show similar endocytic phenotypes. In the macrophage-like cells of vps-45 and rabs-5 mutants, aberrantly small endosomes were accumulated, and the endosome fusion stimulated by the mutant RAB-5 (Q78L) is suppressed by these mutations. Our results indicate that VPS-45 is a key molecule that functions downstream from RAB-5, cooperating with RABS-5, to regulate the dynamics of the endocytic system in multicellular organisms.     

ARF Is Required for Maintenance of Yeast Golgi and Endosome Structure and Function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Δ, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4–64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4–64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.     

Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors

Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network–recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner.