Trafficking to the Apical and Basolateral Membranes In

BRIEF REVIEW www.jasn.org

Trafﬁcking to the Apical and Basolateral Membranes in Polarized Epithelial Cells

Emily H. Stoops and Michael J. Caplan

Departments of Cellular & Molecular Physiology and Cell Biology, Yale University School of Medicine, New Haven, Connecticut

ABSTRACT Renal epithelial cells must maintain distinct protein compositions in their apical and functional residence, proteins can also be basolateral membranes in order to perform their transport functions. The creation of these mis-sorted or randomly delivered and polarized protein distributions depends on sorting signals that designate the traffick- then retained only in the correct compart- ing route and site of ultimate functional residence for each protein. Segregation of ment by interactions with cytoskeletal com- newly synthesized apical and basolateral proteins into distinct carrier vesicles can occur ponents or through other mechanisms that at the trans-Golgi network, recycling endosomes, or a growing assortment of stations effectively immobilize the protein and pre- along the cellular trafficking pathway. The nature of the specific sorting signal and the vent its departure. Additional trafficking mechanism through which it is interpreted can influence the route a protein takes processes provide acute regulation of cell through the cell. Cell type–specific variations in the targeting motifs of a protein, as are surface expression once a protein is local- evident for Na,K-ATPase, demonstrate a remarkable capacity to adapt sorting path- ized to the correct membrane domain. This ways to different developmental states or physiologic requirements. This review sum- is especially true of transport proteins in marizes our current understanding of apical and basolateral trafficking routes in epithelial cells, whose activities are often polarized epithelial cells. tightly controlled in response to stress or

J Am Soc Nephrol 25: 1375–1386, 2014. doi: 10.1681/ASN.2013080883 environmental cues. The topic of transport proteinregulationbytrafficking has been the subject of many reviews.6,7 In the present review, we focus on the biosynthetic Polarized epithelial cells separate an organ- necessary for the generation and mainte- routes pursued by membrane proteins in ’ isms internal milieu from its external envi- nance of the biochemical polarity that is a polarized renal epithelial cells, the mecha- ronment. To perform this barrier function, prerequisite for their physiologic function. nisms that contribute to sorting, and the these cells possess an asymmetric design, Newly synthesized membrane proteins specializations that allow protein sorting “ with an apical membrane facing an out- are packaged into transport vesicles at the pathways to be tailored to a cell’sphysio- ” side lumen and a basolateral membrane trans-Golgi network (TGN) in preparation logic requirements and developmental facing neighboring cells and the basal lam- for their ultimate delivery to the plasma state. ina. These two distinct membrane domains membrane. A protein’s route from the TGN are separated by intercellular junctional to its designated plasma membrane locali- complexes, called tight junctions, which zation can be direct, or it can involve stops BASOLATERAL SORTING SIGNALS render the epithelial cell monolayer selec- at one or more endosomal compart- fl 1–3 tively permeable to solutes and uid. A ments. In addition to the common en- Basolateral sorting of both soluble se- ’ transporting epithelial tissue s functional dosomesthatarepresentinallcelltypes, creted proteins and integral membrane properties are determined in large measure polarized epithelial cells also possess spe- by the inventories of ion channels, trans- cialized endosomal compartments that porters, and pumps that are differentially participate in the trafficking of apical or Published online ahead of print. Publication date localized to their apical and basolateral basolateral proteins, specifically.4,5 The available at www.jasn.org. membranes and that account for the tis- presence of these additional endosomal Correspondence: Dr. Michael J. Caplan, De- sue’s capacity to mediate the net secretion compartments is consistent with the more partment of Cellular and Molecular Physiology, fl fi Yale University School of Medicine, PO Box 208026, or absorption of uid and solutes. Differ- complicated sorting and traf cking pro- New Haven, CT 06520-8026. Email: michael.caplan@ ential sorting and directed targeting of cesses that are required to generate and yale.edu fi membrane proteins to speci cmembrane maintain polarity in these cells. In addition Copyright © 2014 by the American Society of domains in epithelial cells are thus to being targeted directly to their sites of Nephrology

J Am Soc Nephrol 25: 1375–1386, 2014 ISSN : 1046-6673/2507-1375 1375 BRIEF REVIEW www.jasn.org proteins is directed primarily by signals endocytosis. In fact, basolateral signals complexes, mediate the interpretation embedded within the sorted protein’s can overlap with or serve as endocytosis of both tyrosine-based and dileucine tar- primary structure (Table 1). The most signals,8–10,13–16 although this is not al- geting sequences.22–24 The adaptor pro- common types of signals involved in ways the case.15,17,18 Additionally, baso- tein (AP) complexes function as cargo the sorting of basolateral membrane lateral sorting motifs involving a single receptors in the formation of clathrin- proteins are tyrosine-based (NPxY or leucine residue have been identified in coated pits and vesicles.25–31 Tyrosine- YxxØ) or dileucine (D/ExxxLL) mo- several proteins, including stem cell fac- based basolateral signals interact with tifs.8–12 These sequences are embedded tor,19 CD147,20 and amphiregulin m1B, an epithelial-specificisoformof within these proteins’ cytosol-facing do- (EExxxL).21 Protein components of the the m1 subunit of the heterotetrameric mains and are similar in composition cellular trafficking machinery, including AP complex.32–34 Less is known about and geometry to the signals that drive subunits of the clathrin adaptor the nature of signals that drive the targeting of proteins with multiple transmembrane domains, although some of Table 1. Signals and mechanisms for sorting to the apical and basolateral these proteins are known to dimerize membranes with single-pass membrane proteins Presumed Sorting that convey sorting information.35,36 Sorting Signal Protein References Mechanism Basolateral Tyrosine-based Low-density lipoprotein Recycling, but not delivery, 10, 152 APICAL SORTING SIGNALS receptor is m1b-dependent Vesicular stomatitus virus m1b-dependent 153, 154 Apical signals are more diverse than their G protein basolateral counterparts. Topologically, lgp120 m3a-dependent 9, 155 apical signals can reside in any domain of m Dileucine Fc receptor FcRII-B2 1b-independent 11, 15, 156 an apically sorted protein’s structure— Mannose 6-phosphate m1b-independent 157, 158 extracellular, transmembrane, or cyto- receptor E-cadherin Rab11-mediated 3, 12 solic. Compositionally, apical signals Monoleucine Stem cell factor 19 can be based on amino acids, lipids, or CD147 Clathrin-mediated; 130 carbohydrates. The apical membrane is m1b-dependent divided into the ciliary region (which in- Amphiregulin Recycling, but not delivery, 21 cludes the periciliary base and the pri- is m1b-dependent mary cilium itself) and the nonciliary Apical membrane, and these domains are com- GPI-anchor Decay accelerating factor Lipid raft–associated 40, 41 positionally distinct from one another. – Folate receptor Lipid raft associated 43 This division adds further complexity N-Glycans Clusterin (gp80) Raft-independent 46, 159 to the process of apical sorting.37 gp114 Galectin-3–mediated, 47, 61 The recognition that multiple glycosyl- raft-independent Growth hormone Independent of 48 phosphatidylinositol-anchored proteins galectins 3 and 4 (GPI-AP) are localized to the apical mem- Erythropoietin Cholesterol-dependent 49, 160 branes of MDCK cells provided a first clue Endolyn Raft-independent 50 to the existence and nature of apical sort- O-Glycans p75 neurotrophin Galectin-3–mediated, 53, 61 ing signals.38 The sorting behaviors of receptor raft-independent chimeric proteins suggest that the GPI Lactase phlorizin Galectin-3–mediated, 61 anchor is sufficient to specify sorting to hydrolase raft-independent the apical membrane.39,40 However, MUC1 Raft-independent 99, 161, 162 other reports indicate that the anchor is Podocalyxin Transient lipid raft 52 necessary to ensure apical sorting of GPI- association fi 41 – APs, but is not in itself suf cient. The Dipeptidyl peptidase IV Lipid raft associated 55, 58 fi Sucrase isomaltase Lipid raft–associated 55, 56 nding that GPI-APs oligomerize and Transmembrane Neuraminidase Lipid raft–associated 67, 70 partition into detergent-resistant mem- domain branes while trafficking to the apical Influenza hemagglutinin Lipid raft–associated 69, 163 membrane42,43 is consistent with the hy- Respiratory syncytial Lipid raft–associated 71, 164 pothesis that lipid rafts play critical roles virus F protein in apical protein sorting.44,45 Affinity of Sucrase isomaltase Lipid raft–associated 165 GPI-APs for glycosphingolipid-enriched H,K-ATPase Raft-independent 72 rafts is thought to cause their preferential

1376 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1375–1386, 2014 www.jasn.org BRIEF REVIEW incorporation into vesicles bound for the protein folding or inhibit aggregation, The variable outcomes of seemingly apical membrane. thus allowing protein sequence-based sig- straightforward experiments designed to Both N-andO-linked glycosylation nals to interact with protein receptors or test the activitiesof specific types of apical have also been shown to serve as sorting lipid domains.66 signals complicate the establishment of signals for many apical proteins. The po- Transmembrane domain sequences the role of any particular signal or class of tential for N-glycans to act as apical sort- are responsible for the apical sorting of signals in apical sorting. Some of this ing signals was first recognized for the multiple viral single-pass membrane complexity may be attributable to the glycoprotein gp80, which is missorted proteins, including hemagglutinin, existence of redundant or multipart after treatment with N-glycosylation in- neuraminidase, and the respiratory syn- signals. One example of such a bipartite hibitors.46 N-Glycans have since been cytial virus F protein.67–71 A transmem- apical signal is found in podocalyxin. shown to fulfill this function for many brane domain also appears to play a key Individual removal of a heavily glycosy- proteins in studies using glycosylation- role in apical sorting of the polytopic lated region in the ectodomain or of a deficient cell lines or mutagenesis.47–51 gastricH,K-ATPaseprotein.72 The cytosolic region containing a C-terminal Insertion of a site for addition of N- a-subunits of the H,K-ATPase and Na, postsynaptic density 95/disc-large/zona glycans to the sequence of nonglycosy- K-ATPase pumps share approximately occludens domain interaction motif lated rat growth hormone resulted in this 65% sequence identity,73 permitting produced only minimal effects on the protein being secreted apically rather the construction of structurally intact protein’s steady state localization.52 In than without polarity,48 whereas deletion and functionally active chimeric pump contrast, removing both of these apical of one N-glycan chain from erythropoi- proteins. A chimera in which the fourth signal domains resulted in podocalyxin etin impaired apical trafficking.49 Simi- transmembrane domain of the H,K-AT- protein that exhibits an entirely nonpo- larly, chimeric constructs containing the Pase a-subunit was inserted into the larized localization.52 The multiplicity of O-glycosylated region of the glycoprotein complementary position of the basolat- studies exploring the various classes of podocalyxin are endowed with a signal eral Na,K-ATPase a-subunit was targeted apical signals and the roles of lipid rafts for apical targeting,52 and removal of to the apical membrane in transfected ep- clearly demonstrate that apical targeting O-glycans from neurotrophin recep- ithelial cells.72 is not the product of a simple binary tors results in sorting to the basolateral As is the case for GPI-APs, all major “on-or-off switch” process. Further rather than apical membrane.53 As for classes of apical sorting signals have been studies will be required to explore the N-glycans, the importance of O-glycans implicated in inducing clustering of their individual functions of specificsignal for apical trafficking has been demon- associated proteins intodetergent-resistant types, as well as the roles for oligomeri- strated using glycosylation inhibi- membrane domains. The role of cluster- zation and for hierarchical and co- tors.54–58 However, studies on the roles ing in driving biosynthetic apical sorting, operative interplay in determining the of glycans as sorting signals have been however, is murky at best. Mutational mechanisms through which complex complicated by nontargeting effects analysis of the influenza virus hemagglu- signals are interpreted. of their removal by biochemical or ge- tinin (HA) protein transmembrane do- netic means. For example, removal of a mainshowedthatthissequenceisrequired protein’s N-glycans can cause its reten- for the HA protein’s raft association, but SORTING SITE FOR BIOSYNTHETIC tion in the endoplasmic reticulum (ER) not for its apical sorting.74 Furthermore, CARGO due to improper folding.59 Thus, their GPI-APs lose raft association when cells exact role in apical sorting remains are subjected to mild levels of cholesterol To understand the mechanisms mediat- unclear. depletion, but are still sorted to the apical ing trafficking in polarized epithelial Mechanistically, lectin proteins are membrane.75 Secreted forms of GPI-APs cells, it is necessary to elucidate the site postulated to serve as the sorting receptors lacking the GPI linkage are targeted api- at which basolateral proteins and apical that transiently cluster N-glycosylated cally despite having lost both their raft proteins are separated from one another proteins into apically destined do- association and the apical influence pre- as they pursue their postsynthetic traffick- mains.60 Galectins 3, 4, and 9 are lectins viously attributed to GPI.76 A 2005 re- ing itineraries. Several studies address this that have been implicated in apical sorting view of epithelial trafficking suggested issue using biochemical techniques or in kidney and intestinal cells.61–64 The the novel unifying hypothesis that apical microscopy-based assays. By assessing minimal mobility of glycosylated growth signalsfunctionasoligomerization- the sialicacidcontentof the sugarsattached hormone in endosomes, as shown by promoting factors.77 Support for this to the basolaterally directed vesicular sto- fluorescence recovery after photobleach- conjecture is supplied by data showing matitis virus glycoprotein (VSV-G), Fuller ing, is consistent with the postulated role that a GPI-linked green fluorescent pro- et al. demonstrated that this protein re- of lectin tethering in N-glycan–dependent tein (GFP) construct was sorted to the mains in physical contact with the apically sorting.65 Alternative theories suggest that apical membrane unless the GFP con- directed influenza neuraminidase (whose N-glycans serve not as sorting signals but tained mutations known to abolish its enzymatic activity is capable of desialating rather as structural supports that enhance tendency to oligomerize.43 VSV-G) through the late Golgi.78

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Similarly, following infection of MDCK In addition to separation of apical and –independent classes of apical cargo seg- cells with two viruses that bud from oppo- basolateral cargo, distinct sorting of regate from one another by vesicle fis- site membranes—VSV-G from the baso- some proteins bound for the same des- sion following TGN exit.85 Furthermore, lateral membrane and HA from the apical tination has also been observed within as discussed below, a substantial number membrane—Rindler and colleagues ob- the TGN. Basolateral proteins are often of post-Golgi protein sorting processes served colocalization of VSV-G and HA classified according to the dependence of occur in the endosomal system. Finally, within the same Golgi cisternae by colloi- their sorting on the m1B clathrin adaptor some proteins are sorted via a transcel- dal gold immuno-electron microscopy.79 subunit. Two basolateral proteins—the lular route. Although few proteins ap- In contrast, work with GFP-tagged sergly- m1B-dependent VSV-G protein and pear to pursue such pathways in renal cin protein has suggested apical/basolat- m1B-independent Na,K-ATPase—exit epithelial cells, many apical proteins in eral separation as early as the cis Golgi the TGN in separate carriers, demon- hepatic cells are sent first to the basolat- or ER.80,81 Furthermore, studies suggest strating that the role for the TGN in ep- eral membrane, followed by endocytosis the existence of a novel class of detergent- ithelial sorting extends beyond the strict and transport to the apical/canalicular resistant membrane domains that act as apical/basolateral dichotomy.87 m1B is membrane.94 aggregators of apical proteins in the ER.82 expressed in a subset of epithelial tissues Further research will be necessary to de- and, as part of the AP1 clathrin coat termine whether this type of early sepa- adaptor complex, facilitates sorting of SORTING THROUGH ENDOSOMES ration plays an obligate functional role in proteins that use tyrosine-based sorting epithelial protein sorting. signals (such as VSV-G) through the The endosomal system is an essential com- One case of well established early Rab8-positive common recycling endo- ponent of a cell’strafficking network—both segregation involves a specific class of some (CRE).88 As a complement to this endocytic and exocytic—for membrane apical proteins. GPI-APs have been pathway, the TGN-localized m1A iso- proteins, lipids, receptor-bound ligands, shown in yeast to exit the ER in different form was recently shown to facilitate and various solutes (Figure 1). In polarized vesicular carriers than other apical TGN export of a GFP-tagged LDL-R cells, proteins and lipids endocytosed cargo.83 Most recently, the p24 family construct, which contains a nonconven- from the apical and basolateral surface of transmembrane proteins was identi- tional tyrosine-based basolateral signal enter the apical (AEE) and basolateral fied in yeast as the receptor/adaptor for and does not typically traverse the endo- early endosomes, respectively.95 From GPI-APs, facilitating their incorporation somal system.89 there, proteins can be sorted to the sur- into coat protein complex II–coated A nonconventional route for apical face, targeted to the lysosomes for ulti- vesicles in the ER and regulating coat transit that bypasses the Golgi apparatus mate degradation, or transported to the protein complex I–mediated retrograde has also been proposed for a subset of CRE or apical recycling endosome transport of escaped, unremodeled GPI- apical proteins.90,91 AGFP-taggedver- (ARE). The ARE is a cup-shaped com- APs.84 Because GPI-AP signals are sion of the serglycin protein was shown partment in the subapical region of epi- chemically distinct from other sorting to traffic to the apical membrane in thelial cells characterized by the presence signals, which tend to be short amino the presence of the Golgi transport– of the small GTPase Rab11a and the mo- acid sequences or post-translational blocking drug brefeldin.92 Apical de- tor protein Myosin Vb (MyoVb), and the modifications, it is perhaps not surpris- livery was also observed when a KDEL absence of rapidly recycling transferrin ing that their sorting would be mecha- ER retrieval sequence was appended to receptor.96,97 nistically and temporally distinct. serglycin to prevent the protein’s prog- Multiple biosynthetic pathways pass- Despite these examples of early seg- ress along the classic secretory pathway ing through the endosomal system en regation, most sorting appears to occur at from the ER to the Golgi.92 Recently, route to the apical membrane have been some point after proteins have passed analysis of the glycosylation patterns of observed. An elegant demonstration through the late Golgi and reached the newly synthesized polycystin-2 sugges- of trafficking of newly synthesized raft- TGN. Evidence in favor of this argument ted that the ciliary pool of this protein dependent and raft-independent proteins comes from live imaging studies, which exits the Golgi at the cis compartment, through separate endosomal compart- have revealed the separation of proteins rather than from the TGN.93 Additional ments was obtained by functionally in- into different vesicles upon or directly studies focusing on newly synthesized activating the apical early and apical after their export from the TGN.85,86 In cohorts of native proteins expressed at recycling endosomes.98,99 Overexpres- these experiments, progressive lateral wild-type levels in untreated cells will sion of the dominant-negative acting separation of representative apical and be necessary to establish whether there MyoVb-tail domain selectively inhibited basolateral proteins, concurrent with is indeed a Golgi bypass route that func- apical trafficking of the raft-independent the exclusion of resident Golgi proteins tions as a bona fide apical transport apical protein endolyn, while horserad- from these segregated domains, culmi- pathway. ish peroxidase–mediated ablation of the nated in the proteins exiting the Golgi Sorting can also occur after proteins AEE selectively inhibited apical traffick- in distinct carriers. exit the TGN. Lipid raft–dependent and ing of the raft-dependent influenza HA

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protein. Proteins with glycan-dependent signals are thought to traverse the ARE. Glycosylated growth hormone, which re- lies on N-glycans for its sorting, colocalizes with ARE markers en route to the apical surface, and its apical expression is decreased by overexpression of MyoVb- tail.65 In contrast, nonglycosylated GH did not localize to the ARE. In the case of the disaccharidases lactase phlorizin hydrolase and sucrase isomaltase—araft- dependent protein and raft-independent protein, respectively—separation does not occur immediately after release from the TGN, but rather after sequential movement through Rab4-, Rab8-, and Rab11-positive endosomal compartments (or perhaps subcompartments).100 Mounting evidence demonstrates that, in addition to being separated into different endosomal compartments, car- goes are segregated into different subdomains within endosomes and that this lateral segregation facilitates cargo sorting (reviewed by Miaczynska and Zerial101). Using immuno-electron microscopy, Sonnichsen et al. demonstrated separation of resident Rab GTPases into distinct endosomal subdomains. Recycling endosomes exhibited Rab4- and Rab11- positive subdomains, and early endosomes possessed distinct Rab4 and Rab5 subdomains.102 Within early endosomes, recycling cargo segregates to tubular domains, while cargo headed for degradation remains in the spherical portion of the endosome.103 More recently, enrich- ment of recycling b2-adrenergic receptor was observed in tubular microdomains of endosomes that are stabilized by a local actin network.104 This localiza- Figure 1. Apical and basolateral proteins pursue multiple biosynthetic routes to the plasma membrane following their exit from the TGN. This modeldepicts these routes as well tion depended on sorting information as some endocytic pathways. Proteins targeted to the basolateral membrane can traffic embedded within the b2-adrenergic re- directly from the TGN (1),87 or traffic through endosomes (2) before surface delivery (3).88,166 A ceptor sequence, and it took place in similar direct route for apical proteins has not been observed. Endocytosed basolateral different tubules and with different ki- proteins traffic through the basolateral early endosome (BEE, 3) and CRE before recycling or netics than did the bulk sorting of the degradation. Apically directed proteins traverse the apical early endosome (AEE, 4a and b) or rapidly recycling transferrin receptor. In the apical recycling endosome (ARE, 5a and b) before apical delivery. GPI-linked proteins, polarized cells, apical and basolateral influenza HA protein, and other raft-dependent proteins traverse the AEE,98,99 while proteins cargo segregated laterally in recycling fi fi such as endolyn and MUC1, whose traf cking depends on glycan residues, traf c through the endosomes in a phosphoinositide 3- 99,167 fi ARE. Conversely, lactase phlorizin hydrolase and sucrase isomalatase traf c between the kinase–dependent manner.105 This seg- apical early and apical recycling endosomes (6).100 Proteins internalized from the apical regation was not observed when the same membrane to the AEE can traffic to the ARE for recycling back to the surface, or to the CRE and late endosomes if they are targeted for degradation (unlabeled arrows). LE, late endosome; proteins were studied in nonpolarized lys, lysosome; MVB, multivesicular body. cells. To fully establish the extent to which lateral segregation is operative

J Am Soc Nephrol 25: 1375–1386, 2014 Polarized Trafficking 1379 BRIEF REVIEW www.jasn.org and functionally important in sorting, Consistent with the vital role of the receptor protein.128 Interestingly, it will be necessary to couple higher- sodium pump and other transport pro- knockdown experiments in MDCK cells resolution imaging of newly synthesized teins in determining the direction of demonstrated that m1B is required for proteins with innovative assays capable solute flow, these distinct distributions correct recycling of coxsackie and ade- of revealing and perturbing domain of the Na,K-ATPase both reflect and novirus receptor to the basolateral mem- separations within endosomes, the determine the physiologic properties of brane, but not for its initial biosynthetic Golgi network, and earlier trafficking their respective cell types. In renal tubu- delivery.128,129 Absence of m1B cannot compartments. lar epithelial cells, basolateral sodium explain the apical localization of the pump generates the transepithelial so- Na,K-ATPase or the similarly “mis-sorted” dium gradients that drive most of the CD147 in RPE cells, as the trafficking of CELL-SPECIFIC VARIATIONS IN kidney’sabsorptivefluxes of solutes and these proteins is m1B independent. While TRAFFICKING water.121 In the choroid plexus, the api- the underlying mechanism responsible calpoolofNa,K-ATPaseproducesthe for the apical accumulation of CD147 Even when expressed in nonpolarized secretory flux that is necessary for the in RPE remains a mystery, apical cells, apical and basolateral proteins can generation of cerebral spinal fluid.122 CD147 appears to facilitate the apical ac- be separated into different cargo vesi- One possible mechanism for differ- cumulation of another protein, mono- cles.86,106,107 This suggests that the ential localization may relate to ankyrin- carboxylate transporter-1.130 In the case machinery required to accomplish po- fodrin complexes, which have been of the Na,K-ATPase, varying levels of the larized sorting is expressed ubiquitously. demonstrated to help link the Na,K- adherens junction protein E-cadherin However, polarized sorting can manifest ATPase to the cytoskeleton and thus were shown to affect the sodium pump’s highly cell type–specific patterns. The influence its localization. A role for apical versus basolateral localization in multiplicity of signal classes that are in- basolateral ankyrin-fodrin in facilitating RPE cells. RPE cells expressing lower lev- volved in apical and basolateral sorting, Na,K-ATPase basolateral localization has els of E-cadherin exhibit more apical Na, as well as the complex array of vesicular been suggested in MDCK and renal K-ATPase than do cells possessing higher compartments that participate in the tubule cells.123,124 Conversely, ankyrin E-cadherin levels.131 The level of ankyrin segregation of apical and basolateral and fodrin were both found at the apical and fodrin expression did not correlate proteins bearing these signals, allows surface of choroid plexus and RPE cells, with Na,K-ATPase localization in these for a plastic system in which distinct sub- where ankyrin directly interacts with Na, cells. sets of proteins can be redirected in re- K-ATPase.125,126 Fodrin and ankyrin sponse to environmental cues. Several also localize to the lateral membrane of Developmental Changes in Sorting proteins show cell-specificvariationsin choroid plexus cells, however,125 sug- Studies performed on fetal kidneys re- trafficking.108–112 To demonstrate the gesting that the presence of these cyto- vealed the presence of the Na,K-ATPase remarkable capacity of the protein traf- skeletal linkers is not sufficient to specify a1-subunit at the apical and lateral ficking system to adapt to the physio- Na,K-ATPase targeting. It is more likely membranes (or distributed in a nonpo- logic requirements of particular cell that this cytoskeletal attachment plays a larized manner) of renal vesicles and states and tissues, we will focus on cell role in retention at the membrane, newly formed collecting ducts in early types in which the Na,K-ATPase com- rather than directly determining the stages of kidney organogenesis (Figure plex exhibits an apical localization. As pump’s initial trafficking or ultimate dis- 2).132 This change in localization of ac- previously mentioned, the Na,K-ATPase tribution. It will be informative to more tive sodium pump is thought to play an complex localizes to the basolateral closely follow the biosynthetic route of important role in the formation of the membrane of most polarized cells, native Na,K-ATPase protein in both re- lumen during tubulogenesis.133 Burrow where it plays an important role in the nal and choroid plexus epithelial cells to and colleagues further showed that the regulation of ion transport. parse out the alternate roles of selective timing of the postnatal switch to baso- targeting and selective retention in cre- lateral a1 distribution closely mirrored a Cell Type–Specific Variations in ating the distinct localizations of Na,K- switch in the expression of b-subunit Sorting ATPase these cell types exhibit. Recent isoforms. The sodium pump consists of The Na,K-ATPase localizes to the apical advances in the generation of an immor- two obligate subunits (a and b), as well membranes of cells of the retinal pigment talized choroid plexus cell line for use in as a regulatory component (g). The cat- epithelium (RPE)113–116 and the choroid in vitro experiments may prove ex- alytic a-subunit must assemble with the plexus.117–120 Although these cells exhibit tremely valuable in efforts to understand heavily glycosylated b-subunit in the ER an apical distribution of this canonical Na,K-ATPase trafficking in this tissue.127 in order to become catalytically active and basolateral protein, other proteins that In the case of RPE cells, the absence of to reach the cell surface.36,134–136 There serve as standard apical and basolateral epithelial-specific m1B AP expression is are three a-subunit and two b-subunit markers retain their characteristic dis- associated with the aberrant apical local- isoforms; in mature tubules, the predom- tributions. ization of the coxsackie and adenovirus inant heterodimer is a1b1. While b2was

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Figure 2. Na,K-ATPase displays distinct polarized localization in different tissues and developmental stages. (A–C) Immunolocalization of sodium pump a-subunit in murine choroid plexus (A), developing kidney (B), and adult kidney (C). Arrowheads indicate apical localization of a-subunit. Arrows indicate basolateral localization of a-subunit. (A) Sodium pump a-subunit localizes to the apical membrane of choroid plexus cells. (B) In developing kidneys harvested from embryonic day 17 mice, sodium pump a-subunit localizes to the apical membrane of the epithelial cells of the renal vesicles, which are the earliest structures in nephrogenesis. In newly developing collecting ducts, sodium pump a-subunit localizes to both apical and basolateral membranes or—in more mature collecting ducts—to basolateral membranes only. (C) Sodium pump a-subunit localizes to the basolateral infoldings of adult kidney tubules. (D–F) Schematic views of sodium pump localization in choroid plexus (D), the developing kidney (E), and the adult kidney (F). Sodium pump a-subunit is depicted in red. Bar=10 mm. expressed in the developing kidney, b1 for these behaviors remain largely un- In Dent disease, mutations in the chloride protein was not observed, despite the characterized. Clearly, however, while channel ClC-5 result in redistribution presence of equivalent levels of b1 and much of the polarized sorting machin- of H+-ATPase to the basolateral mem- b2mRNA.133 b2 is also expressed in ery is present in all cell types, the regu- brane rather than the apical membrane RPE,137 in the choroid plexus,138 and in lated expression of distinct effector of proximal tubule cells.141,142 The ClC- tissue from patients with autosomal proteins can play a role in fine-tuning 5 mutation also impairs the function of dominant polycystic kidney disease,139 protein trafficking to a cell’sspecific the endocytic system, contributing to pro- where apical localization of Na,K-ATPase physiology and developmental state. teinuria. Bartter syndrome can be caused has been observed. These data, combined by mutations that perturb the trafficking with the virtual lack of endogenous b2 or the distribution of the ClC-Kb channel expression in the adult kidney, suggest a IMPAIRED PROTEIN TRAFFICKING or the renal outer medullary K+ chan- role for b2 in guiding the apical accumu- AND DISEASE nel.143 Mutations resulting in decreased lation of the Na,K-ATPase. The mecha- internalization and downregulation of nism through which the b2-subunit Perturbations in the complicated network the epithelial sodium channel cause Liddle might exert these effects remains to be of subcellular trafficking pathways are syndrome.144 The resulting constitutive determined. implicated in many diseases.140 Patho- activity of the epithelial sodium channel Epithelial cells can manifest tissue genic defects in trafficking include in- at the apical membrane leads to excessive type–specific or developmental stage– verted polarity, disrupted recycling and sodium retention and pseudohyperal- dependent idiosyncrasies in their sorting intracellular retention, as highlighted in dosteronism.145 In the case of nephro- behaviors. The mechanisms responsible the following examples of renal pathology. genic diabetes insipidus, mutations in the

J Am Soc Nephrol 25: 1375–1386, 2014 Polarized Trafficking 1381 BRIEF REVIEW www.jasn.org gene encoding the collecting duct wa- Fellowship Grant No. DGE-1122492 (E.H.S.) polarized delivery of influenza virus hem- – ter channel aquaporin-2 can produce and National Institutes of Health grants agglutinin. JCellBiol114: 413 421, 1991 14. Prill V, Lehmann L, von Figura K, Peters C: DK17433 and DK072612 (M.J.C.). a functional but misrouted channel The cytoplasmic tail of lysosomal acid that fails to reach the plasma mem- phosphatase contains overlapping but dis- brane.146,147 Without aquaporin-2 at tinct signals for basolateral sorting and REFERENCES the apical membrane, the water perme- rapid internalization in polarized MDCK – ability of collecting tubule epithelial cells cells. EMBO J 12: 2181 2193, 1993 1. Futter CE, Connolly CN, Cutler DF, Hopkins 15. 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