Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.     

Rab14 regulates apical targeting in polarized epithelial cells

Epithelial cells display distinct apical and basolateral membrane domains, and maintenance of this asymmetry is essential to the function of epithelial tissues. Polarized delivery of apical and basolateral membrane proteins from the trans Golgi network (TGN) and/or endosomes to the correct domain requires specific cytoplasmic machinery to control the sorting, budding and fission of vesicles. However, the molecular machinery that regulates polarized delivery of apical proteins remains poorly understood. In this study, we show that the small guanosine triphosphatase Rab14 is involved in the apical targeting pathway. Using yeast two-hybrid analysis and glutathione S-transferase pull down, we show that Rab14 interacts with apical membrane proteins and localizes to the TGN and apical endosomes. Overexpression of the GDP mutant form of Rab14 (S25N) induces an enlargement of the TGN and vesicle accumulation around Golgi membranes. Moreover, expression of Rab14-S25N results in mislocalization of the apical raft-associated protein vasoactive intestinal peptide/MAL to the basolateral domain but does not disrupt basolateral targeting or recycling. These data suggest that Rab14 specifically regulates delivery of cargo from the TGN to the apical domain.     

The Large GTPase Mx1 Is Involved in Apical Transport in MDCK Cells

In epithelial cells apical proteins are transported by specific transport carriers to the correct membrane domain. The composition of these carriers is heterogeneous and comprises components such as motor proteins, annexins, lectins, Rab GTPases and cargo molecules. Here, we provide biochemical and fluorescence microscopic data to show that the dynamin‐related large GTPase Mx1 is a component of post‐Golgi vesicles carrying the neurotrophin receptor p75^NTR. Moreover, siRNA‐mediated depletion of Mx1 significantly decreased the transport efficiency of apical proteins in MDCK cells. In conclusion, Mx1 plays a crucial role in the delivery of cargo molecules to the apical membrane of epithelial cells.      

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

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.     

Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti

Two-electrode voltage clamp (TEVC) methods were used to explore conductive transport pathways in principal cells, the dominant cell type in Malpighian tubules of the yellow fever mosquito. The basolateral membrane of principal cells had a voltage (V_bl) of -85.1 mV in 49 principal cells under control conditions. Measures of the input resistance R_pc together with membrane fractional resistance yielded estimates of the conductance of the basolateral membrane (g_bl = 1.48 μS) and the apical membrane (g_a = 3.13 μS). K⁺ channels blocked by barium accounted for 0.94 μS of g_bl. Estimates of transference numbers yielded the basolateral membrane Na⁺ conductance of 0.24 μS, leaving 0.30 μS (20%) of g_bl unaccounted. The secretagogue db-cAMP (0.1 mM), a known activator of the basolateral membrane Na⁺ conductance, significantly depolarized V_bl to -65.0 mV and significantly increased g_bl from 1.48 μS to 2.47 μS. The increase was blocked with amiloride (1 mM), a known blocker of epithelial Na⁺ transport. The inhibition of metabolism with di-nitrophenol significantly depolarized V_bl to -9.7 mV and significantly increased R_pc from 391.6 kΩ to 2612.5 kΩ. Similar results were obtained with cyanide, but it remains unclear whether the large increases in R_pc stem from the uncoupling of epithelial cells and/or the shutdown of conductive transport pathways in basolateral and apical membranes. Our results indicate that the apical membrane of principal cells is more than twice as conductive as the basolateral membrane. Partial ionic conductances suggest the rate-limiting step for transepithelial Na⁺ secretion at the basolateral membrane.     

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.     

A tale of three GTPases and a RIN in endothelial cell adhesion

Endothelial cell adhesion to the extracellular matrix regulates migration and outgrowth of blood vessels during angiogenesis. Cell adhesion is mediated by integrins, which transduce signals from the extracellular environment into the cell and, in turn, are regulated by intracellular signaling molecules. In a paper recently published in Cell Research, Sandri et al. show that RIN2 connects three GTPases, R-Ras, Rab5 and Rac1, to promote endothelial cell adhesion through the regulation of integrin internalization and Rac1 activation.The formation of the vascular tree during development requires the orderly growth of blood vessels to irrigate all organs and tissues. This process of blood vessel remodeling, termed angiogenesis, requires endothelial cell proliferation, adhesion, migration and tube formation¹. Pathological angiogenesis takes place during tumor growth as hypoxia within the tumor induces the release of pro-angiogenic mediators such as vascular endothelial growth factor (VEGF).Small GTPases are critical for the regulation of cell behavior and thus also play a central role in angiogenesis. Small GTPases are 20-25 kDa signaling proteins that cycle between an active GTP-bound and an inactive GDP-bound state. When active, GTPases associate with and activate diverse effector molecules that subsequently relay the signal to other molecules, ultimately leading to a specific cell response. Two classes of proteins facilitate GTPase cycling. Guanine exchange factors (GEFs) catalyze GDP unloading thereby promoting GTP binding and GTPase activation. Conversely, GTPase activating proteins (GAPs) enhance the intrinsic GTP hydrolysis activity of the GTPase leading to its inactivation. Small GTPases form a large superfamily with over 100 members in mammals. Based on structural and functional criteria, the GTPase superfamily is subdivided in Ras, Rab, Rho, Arf and Ran subfamilies, each of them generally, but not exclusively, specialized in the regulation of specific cellular events. For example, Rho GTPases primarily regulate cytoskeletal dynamics; Rab GTPases regulate intracellular membrane trafficking; and Ras GTPases function in the regulation of cell proliferation and survival. However, complex processes such as angiogenesis require the coordinated action of several GTPases. This is evidenced by the work of Sandri et al.² recently published in Cell Research. In their paper, Sandri et al. propose a mechanism for the regulation of endothelial cell adhesion and migration involving three GTPases belonging to different GTPase branches, R-Ras, Rab5 and Rac1. The protein RIN2 (Ras and Rab adaptor 2) brings together R-Ras and Rab5 to form a signaling module that orchestrates integrin trafficking and Rac1 activation, processes that are essential for cell adhesion and migration.Integrins are heterodimeric transmembrane extracellular matrix (ECM) receptors composed of one α and one β chain. In a process known as ''outside-in'' signaling, integrins transmit signals from the extracellular environment to intracellular adaptor and signaling molecules that regulate cell migration, survival and growth. Conversely, during ''inside-out'' signaling, integrins can be switched from an inactive to an active conformation by cytoplasmic signaling molecules leading to increased integrin affinity for the ECM.During 2D migration of adherent cells, nascent, highly dynamic focal contacts are formed at the leading edge lamellipodia where integrins mediate adhesion to the ECM. Some of these focal contacts disassemble and some mature into larger focal adhesions with a longer half-life. Failure in maintaining a dynamic assembly and disassembly of focal contacts will result in the inhibition of cell migration.Integrin-mediated adhesion can be regulated at different levels: (1) by changing integrin conformation and thus affinity for their ligand; (2) by modulating integrin avidity, i.e., by promoting integrin clustering on the plasma membrane; and (3) by changing the kinetics of integrin endocytosis and/or recycling³.The Ras GTPase R-Ras is primarily expressed in the vascular system (endothelial cells and vascular smooth muscle cells)⁴. Zhang et al.⁵ were the first to show that R-Ras is a potent regulator of cell adhesion when they reported that expression of active R-Ras was enough to induce ECM adhesion of suspension cells, whereas dominant negative R-Ras reduced adhesion of the adherent cell line CHO. Although R-Ras was shown to enhance integrin affinity⁵, this effect was not consistently observed^6,7. These contradictory findings could be explained by the fact that R-Ras may activate integrins indirectly through antagonizing H-Ras-mediated integrin inhibition⁶.Recent findings suggest that R-Ras stimulates adhesion through the regulation of integrin internalization into Rab11-positive endosomes⁸. Now, the data of Sandri et al.² support this model. The authors addressed the question on how R-Ras regulates cell adhesion of endothelial cells by performing a yeast-two-hybrid screen using constitutively-active R-Ras as bait. The screen revealed that RIN2 is a major R-Ras-interacting protein. RIN proteins (RIN1, 2 and 3) are downstream effectors of Ras GTPases that function as GEFs for Rab5⁹, a GTPase that regulates endocytosis. RIN1 was shown to mediate the stimulation of EGF receptor-mediated endocytosis by H-Ras through the activation of Rab5¹⁰. Surprisingly, Sandri et al. found that R-Ras dramatically impaired the Rab5 exchange activity of RIN2, while H-Ras had no effect. However, RIN2 was still able to specifically bind active Rab5. These data suggest that active R-Ras, RIN2 and active Rab5 form a signaling complex. Accordingly, Sandri et al. show that endogenous R-Ras, RIN2 and Rab5 are indeed found in a complex in endothelial cells. While active R-Ras and RIN2 colocalize at nascent focal contacts and on intracellular vesicles, colocalization with Rab5 takes place on endosomes. The deletion of either the Ras- or the Rab5-binding domains of RIN2 prevented the colocalization of the trio. Thus, RIN2 appears to facilitate the transport of active R-Ras to Rab5-positive endosomes. What is the functional relevance of these interactions? Sandri et al. show that silencing of endogenous RIN2 impaired the increase in adhesion induced by active R-Ras and by Rab5. A similar effect was obtained upon expression of RIN2 deletion mutants lacking Ras- or Rab5-binding domains. These data strongly suggest that the adaptor function of RIN2 in connecting R-Ras and Rab5 regulates endothelial cell adhesion to the ECM. But what is the mechanism? Previous work has shown that the pro-adhesive activity of active R-Ras is linked to its ability to regulate β1 integrin endocytosis⁸. Sandri et al. confirm these data by showing that silencing of R-Ras or RIN2 decreases the rate of endocytosis of active ECM-engaged β1 integrins. In addition, the authors set a step further as they show that the signaling complex R-Ras/RIN2/Rab5 mediates basal Rac1 GTPase activation. Rac1 regulates actin dynamics and ruffle formation at the leading edge of migrating cells and its activity is essential for cell adhesion and migration. TIAM-1-mediated activation of Rac1 on endosomes and subsequent polarized transport to the plasma membrane has been proposed as a way to restrict Rac activity to sites of membrane protrusion^11,12. In line with this model, Sandri et al. show that active R-Ras and RIN2 colocalize with Rac1 on endosomes and that the endosomal Rac GEF TIAM-1 is necessary for R-Ras- and RIN2-induced cell adhesion.Altogether, the data of Sandri et al. support a model in which, integrin-activated R-Ras recruits RIN2 to focal adhesions and induces RIN2 conversion from a Rab5 GEF to a Rab5-docking protein. Subsequently, the complex promotes the endocytosis of ECM-engaged integrins and moves to early endosomes where R-Ras activates the TIAM-1/Rac1 pathway¹³. Active Rac1 translocates to the plasma membrane where it promotes actin polymerization and formation of new focal contacts (Figure 1).Open in a separate windowFigure 1Model proposed by Sandri et al.² for the regulation of focal adhesion dynamics by R-Ras. (1) R-Ras is activated by ECM-engaged integrins, recruits RIN2 and converts it from a Rab5 GEF to a Rab5 adaptor; (2) RIN2 binding to active Rab5 mediates the endocytosis of integrins and the transport of active R-Ras to endosomes; (3) R-Ras contributes to the activation of the Rac1 GEF TIAM-1, which then activates Rac1; (4) Active Rac1 translocates to the plasma membrane and promotes actin polymerization and formation of new focal contacts.By bridging active R-Ras and Rab5, RIN2 combines two processes essential for cell adhesion: (1) focal contact dynamics through the internalization of ECM-engaged integrins; and (2) local Rac1 activation to ensure actin polymerization at lamellipodia. Similarly, RIN2 also connects H-Ras and Rab5 in the internalization of the epithelial cell-cell adhesion molecule E-cadherin¹⁴. Thus, RIN2 appears to be a universal effector of Ras-induced endocytosis of membrane receptors.Interestingly, the phenotype of a family with a homozygous mutation in RIN2 was recently described¹⁵. The affected individuals showed diverse abnormalities related to a defective connective tissue. Indeed, ultrastructural analysis of the skin showed an abnormal morphology of collagen fibrils. Collagen is a ligand for β1 integrins. Through simultaneous binding to collagen and to the intracellular cytoskeleton, integrins contribute to the assembly of the ECM by transmitting contraction forces from the cell to the ECM. It is tempting to speculate that the phenotype of the patients lacking RIN2 is due to a deficient β1 integrin function as found by Sandri et al. in their in vitro analysis. In addition, these patients bruise easily and present prolonged bleeding, which could be caused by deficient wound healing of blood vessels as a consequence of impaired R-Ras signaling.It should be noted, however, that R-Ras knockout mice have no major defects in vascular development but respond with increased angiogenesis to stress conditions such as tumor implantation⁴. On the contrary, the in vitro study by Sandri et al. suggests that R-Ras deficiency results in decreased endothelial cell migration. Further research is needed to clarify the role of R-Ras in angiogenesis. Likewise, it will be interesting to study vascular responses in RIN2-deficient mice in comparison to R-Ras knockout mice.     

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain–binding protein 1–like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1–dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.     

Apico-basal polarity in polycystic kidney disease epithelia

Epithelial cell polarity is essential for the establishment and maintenance of morphological and functional asymmetries that underlie normal renal structure and function and are brought about by the appropriate delivery of growth factor receptors and ion and fluid transporters and channels to apical or basolateral cell membranes. The fundamental process of cellular polarization is established early during development and is controlled by sets of evolutionarily conserved proteins that integrate intrinsic and extrinsic polarity cues. Specialized structural domains between adjacent cells and cells with their matrix, termed adherens junctions (AJ) and focal adhesions (FA), respectively, are formed that contain specific components of multi-molecular complexes acting as sites to recruit proteins and to activate intracellular mechano-transduction pathways. Regulation of these processes results in tight spatio-temporal control of renal tubule growth and lumen diameter. Abnormalities in macromolecular polarization complexes lead to a variety of diseases in different organs, a common example of which is Polycystic Kidney Disease (PKD), where epithelial cysts replace normal renal tubules. Membrane protein polarity defects in Autosomal Dominant (AD) PKD include the mis-polarization of normally basolateral membrane proteins to apical, lumenal membranes, such as epidermal growth factor (EGFR/ErbB) receptors and Na⁺K⁺-ATPase-α1 subunit; mis-polarization of normally apical membrane proteins to basolateral membranes, including the Na⁺K⁺2Cl⁻ (NKCC1) symporter; and the failure to traffic and insert proteins into membranes resulting in their intracellular accumulation, such as E-cadherin and the β1 subunit of Na⁺K⁺-ATPase. Abnormalities in structural AJ, FA and polarity complexes in ADPKD epithelia include loss of E-cadherin, and focal adhesion kinase (FAK), MALS-3, Crb and Dlg complexes as well as disruptions in Rab/sec and syntaxin trafficking and membrane docking pathways. Since proper polarization of epithelial cells lining renal tubules is essential for normal kidney development and differentiation to prevent abnormal cystic dilation, interventions to reverse polarity defects to normal would offer therapeutic opportunities for PKD. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

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.     

Rab6 functions in polarized transport in Drosophila photoreceptors

Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected.

The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs.     

Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity

Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR?proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.     

Expression of transfected human Na+/H+ exchanger (NHE-1) in the basolateral membrane of opossum kidney cells

Some epithelial cells have Na⁺/H⁺ exchanger (NHE) activity in both apical and basolateral membranes. Amiloride-sensitive NHE-1 is generally identified in the basolateral membrane. The renal cell line, OK7a, targets amiloride-resistant NHE predominantly to the apical membrane. It is controversial whether the transfected NHE-1 is targeted preferentially to the basolateral membrane in OK7a cells, when human NHE-1 is chronically expressed under control of constitutively active promoters. We tried to identify the membranes in which the transfected human NHE-1 could be detected following acute expression in OK7a cells. We have always observed small Na⁺-dependent pH recovery in the basolateral membrane in OK7a cells. It is, however, controversial whether or not OK7a cells express NHE activity in the basolateral membrane. We also characterized Na⁺-dependent pH recovery in the basolateral membrane. It was not inhibited by [4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid] (DIDS), [4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid] (SITS), or contralateral amiloride. Li⁺ but not K⁺, chol⁺, or NMG⁺ could replace Na⁺. These results are consistent with the presence of the NHE in the basolateral membrane. NHE activities were predominant in the apical membrane and those in both membranes were resistant to amiloride analogs. After stable transfection with human NHE-1 in a vector utilizing the metallothionein promoter, overnight induction with Zn²⁺ increased the NHE activity and its sensitivity to amiloride only in the basolateral membrane in OK7a cells. We conclude that the transfected human NHE-1 is exclusively targeted to the basolateral membrane of OK7a cells during acute induction. J Cell Physiol 178:44–50, 1999. © 1999 Wiley-Liss, Inc.     

Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella‐containing vacuoles from macrophages

The opportunistic pathogen Legionella pneumophila employs the Icm/Dot type IV secretion system and ～300 different effector proteins to replicate in macrophages and amoebae in a distinct ‘Legionella‐containing vacuole’ (LCV). LCVs from infected RAW 264.7 macrophages were enriched by immuno‐affinity separation and density gradient centrifugation, using an antibody against the L. pneumophila effector SidC, which specifically binds to the phosphoinositide PtdIns(4)P on the pathogen vacuole membrane. The proteome of purified LCVs was determined by mass spectro‐metry (data are available via ProteomeXchange with identifier PXD000647). The proteomics analysis revealed more than 1150 host proteins, including 13 small GTPases of the Rab family. Using fluorescence microscopy, 6 novel Rab proteins were confirmed to localize on pathogen vacuoles harbouring wild‐type but not ΔicmT mutant L. pneumophila. Individual depletion of 20 GTPases by RNA interference indicated that endocytic GTPases (Rab5a, Rab14 and Rab21) restrict intracellular growth of L. pneumophila, whereas secretory GTPases (Rab8a, Rab10 and Rab32) implicated in Golgi‐endosome trafficking promote bacterial replication. Upon silencing of Rab21 or Rab32, fewer LCVs stained positive for Rab4 or Rab9, implicated in secretory or retrograde trafficking respectively. Moreover, depletion of Rab8a, Rab14 or Rab21 significantly decreased the number of SidC‐positive LCVs, suggesting that PtdIns(4)P is reduced under these conditions. L. pneumophila proteins identified in purified LCVs included proteins putatively implicated in phosphorus metabolism and as many as 60 Icm/Dot‐translocated effectors, which are likely required early during infection. Taken together, the phagocyte and Legionella proteomes of purified LCVs lay the foundation for further hypothesis‐driven investigations of the complex process of pathogen vacuole formation.     

The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin''s cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.     

Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization

The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell- specific small GTPase. Northern blot analysis on various mouse organs, revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport.     

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.     

The Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in polarized Madin-Darby canine kidney cells

The AP-1B clathrin adaptor complex plays a key role in the recognition and intracellular transport of many membrane proteins destined for the basolateral surface of epithelial cells. However, little is known about other components that act in conjunction with AP-1B. We found that the Rab8 GTPase is one such component. Expression of a constitutively activated GTP hydrolysis mutant selectively inhibited basolateral (but not apical) transport of newly synthesized membrane proteins. Moreover, the effects were limited to AP-1B-dependent basolateral cargo; basolateral transport of proteins containing dileucine targeting motifs that do not interact with AP-1B were targeted normally despite overexpression of mutant Rab8. Similar results were obtained for a dominant-negative allele of the Rho GTPase Cdc42, previously implicated in basolateral transport but now shown to be selective for the AP-1B pathway. Rab8-GFP was localized to membranes in the TGN-recycling endosome, together with AP-1B complexes and the closely related but ubiquitously expressed AP-1A complex. However, expression of active Rab8 caused a selective dissociation of AP-1B complexes, reflecting the specificity of Rab8 for AP-1B-dependent transport.     

Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells

Several isoforms of Na⁺/H⁺ exchanger (NHE-1–5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na⁺-dependent pH recovery of the apical and basolateral membranes separately. Na⁺ applied to the apical membrane recovered cell pH. Na⁺-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li⁺ but not K⁺, chol⁺, or NMG⁺ could replace Na⁺. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5′ terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateralmembranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane. J. Cell. Physiol. 171:318–324, 1997. © 1997 Wiley-Liss, Inc.     

Crystal structure of a Chlamydomonas reinhardtii flagellar RabGAP TBC‐domain at 1.8 Å resolution

Rab GTPases play a crucial role in the regulation of many intracellular membrane trafficking pathways including endocytosis and ciliogenesis. Rab GTPase activating proteins (RabGAPs) increase the GTP hydrolysis rate of Rab GTPases and turn them into guanine nucleotide diphosphate (GDP) bound inactive form. Here, we determined the crystal structure of the putative catalytic domain of a RabGAP (which we name CrfRabGAP) that is found in the flagellar proteome of the unicellular green alga Chlamydomonas reinhardtii. BLAST searches revealed potential human orthologues of CrfRabGAP as TBC1D3 and TBC1D26. Sequence and structural comparison with other canonical RabGAPs revealed that the CrfRabGAP does not contain the canonical catalytic residues required for the activation of Rab GTPases. The function of noncanonical RabGAPs‐like CrfRabGAP might be to serve as Rab effectors rather than activators. Proteins 2014; 82:2282–2287. © 2014 Wiley Periodicals, Inc.