Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

Chapter 57 Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells Bernardo Ortega and Paul A. Welling 57.1  INTRODUCTION TO EPITHELIA...

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Chapter 57

Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells Bernardo Ortega and Paul A. Welling

57.1  INTRODUCTION TO EPITHELIAL CELL POLARITY The ability of epithelial cells in the gastrointestinal (GI) tract to absorb selected nutrients while secreting electrolytes, enzymes, and digestive factors depends on intracellular trafficking mechanisms that establish and maintain the polarized distribution of different transport proteins and surface receptors on the apical and basolateral membranes. A combination of intracellular sorting operations, vectorial delivery mechanisms, and plasmalemma-specific fusion and retention processes are responsible. Well-defined signals that specify polarized sorting or retention of proteins have been described, but the intracellular machinery that decodes and acts on these signals is still only partially understood. In this chapter we provide an up-to-date review of the trafficking routes, molecular machinery, and mechanisms involved in protein sorting and trafficking.

57.2  CYTOARCHITECTURE AND MEMBRANE COMPARTMENTS IN POLARIZED EPITHELIAL CELLS Epithelial cells exhibit tremendous diversity and specialization along the GI tract, yet they share many common morphological features. Among those common features are a basement membrane, a complex set of cell junctions that delimits an apical and a basolateral membrane, a cytoskeleton with a specific spatial distribution, and a set of membrane compartments that serve as platforms for polarized sorting and delivery of apical and basolateral membrane proteins. Although a detailed description of these structures exceeds the purpose of this chapter, a brief summary of their peculiar distribution and functional significance is highlighted to provide a context for understanding the mechanisms of membrane polarity (see Figure 57.1).

57.2.1  Epithelial Cell Junctions The tight junction complex at the apex of the cell forms a relatively impermeable barrier between the solutions

on either side of the epithelium and acts as a molecular “fence” to partition the contents of the apical and basolateral membranes from each other.1–3 It is formed of integral membrane proteins, including Occludin, the claudins, and the junction adhesion molecule (JAM), which assemble as a network of strands and interact on opposing membranes of adjacent cells though their extracellular domains.3–5 A number of regulatory and scaffolding proteins associate with the cytoplasmic surface of the tight junction, including ZO-1, -2, and -3, cingulin, 7H6, and symplekin, providing a means to connect the junction to the actin cytoskeleton. They also allow recruitment of signal transduction proteins and polarity-generating complexes,6 creating de facto signaling platforms.7 Importantly, components of the exocyst, a multiprotein complex involved in polarized protein trafficking, also associate with or near tight junctions.7,8 The adhesion between epithelial cells is mostly due to the zonula adherens, located as a belt that encircles the cell just below the tight junctions. Cadherins are the main component of the zonula adherens, with their extracellular domains likely mediating a calcium-dependent transdimerization process that provides adhesion between neighboring epithelial cells. In all, the apical junctional complexes are dynamic structures that undergo dramatic rearrangement and redistribution during embryonic development, cell migration, and proliferation, closely integrating these processes to an equally dynamic cytoskeleton.3 In addition to the apical junctional complexes, desmosomes, composed of desmosomal cadherins, associate to intermediate filament attachment proteins and provide additional anchoring points throughout the basolateral membranes of neighboring epithelial cells, which contribute to the maintenance of the epithelium integrity.9

57.2.2  Cytoskeleton in Epithelial Cells The cytoskeleton in epithelial cells is closely associated with both apical and basolateral membranes, playing important structural and functional roles in protein sorting

Physiology of the Gastrointestinal Tract, Two Volume Set. DOI: 10.1016/B978-0-12-382026-6.00057-9 © 2012 Elsevier Inc. All rights reserved.

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Tight Junction Adherens Junction

Desmosome

Microtubules Microfilaments Intermediate filaments

FIGURE 57.1  Schematic diagram of the main elements conforming the cytoskeleton in a prototypical polarized epithelial cell.  The Golgi has an apical position in polarized cells. Microtubules run along the apical–basal axis of the cell, with their minus-ends anchored in multiple apical non-centrosomal organizing centers (MTOC). In addition, short, randomly oriented MT underlie both apical and basolateral membranes. A dense band of actin microfilaments lies in close proximity with the apical junctional complexes and is important for their regulation. Actin is also located at the core of the apical microvilli in a cortical meshwork of actin that underlies the entire plasma membrane. Intermediate filaments composed of keratin are important to stabilize MT in proximity to the plasma membrane. In addition, keratin microfilaments associate with desmosomes and are important in maintaining the epithelial integrity.

and trafficking. The building blocks of the cytoskeleton — namely microfilaments (MF), microtubules (MT), and intermediate filaments (IF) — are organized as depicted in Figure 57.1 (reviewed in 10). MFs are formed by the polymerization β- and γ-actin monomers, with a fastgrowing plus-end (“barbed”) and slow-growing minus-end (“pointed”). Multiple MFs associate to form filamentous networks for which some cross-linking proteins, such as α-actinin or filamin, are required. In addition, other actinbinding proteins control polymerization dynamics, bundling, nucleation, branching, actin-membrane interaction, cell–extracellular matrix (ECM) interaction, contractility, scaffolding, and signaling.10,11 Microtubules are composed of α- and β-tubulin monomers that combine to produce αβ-dimers prior to being added in a head-to-tail fashion to the fast growing plus-end of an existing MT. This intrinsic polarity is of great importance in defining the directionality of their transport capabilities. In epithelial cells (such as Caco-2, MDCK, or enterocytes) MTs emanate from multiple non-centrosomal organizing centers (MTOC) located below the apical membrane (pericanalicular region in hepatocytes),12 and assemble into polarized bundles running along the apical-to-basolateral axis with their rapidly changing plus-ends projecting toward the basal

membrane.13–15 This classical view of the MT subcellular organization has been recently challenged by the discovery of a group of MTs oriented with their plus-ends toward the apical membrane,16 which appears to play an important role in polarized vesicle transport in MDCK cells (see Section 57.7.1). Intermediate filaments in intestinal epithelial cells are composed mostly of keratin types 8, 18, and 19 and have important structural roles.10 At the apex of the lateral membranes, running along the cell-adhesion junction, densely packed antiparallel arrays of actin filaments associate to myosin II in order to generate a contractile ring. This structure is closely associated with the apical junctional complexes via E-cadherin interactions, and generates an extensive transcellular network important, for example, in canalicular contraction during bile secretion. Underlying the apical membrane, a meshwork of parallel arrays of actin filaments cross-linked by myosin and fodrin provides a physical structure to anchor secretory vesicles in proximity to the plasma membrane during epithelial cell secretion and exocytosis.10,17 A set of randomly organized MTs, with their minus-ends and multiple MTOC embedded in a meshwork of cytokeratins, runs parallel to the apical (and basolateral) domain.13,18 Subapical intermediate filaments also play

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Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

an important role in maintaining cell integrity in digestive epithelia.10 Intestinal microvilli emerging from the apical membrane are supported by the apical actin meshwork and at their cores contain well-organized actin MFs bundled by actin-binding proteins including villin, fimbrin, and small amounts of espin.10 The basolateral membrane is also lined by a meshwork composed of cross-linked arrays of actin. Focal adhesions connect this actin meshwork to the ECM through integrins and their associated cytosolic actinbinding proteins.10,19 In addition, in the small intestine, keratin-associated proteins like polycystin-1 associate with both desmosomes and keratin filaments to establish cell–cell interactions.20 MTs and their motors (dynein and kinesins) are important in vesicle trafficking as well as in organizing and positioning various organelles, such as Golgi, endosomes, and lysosomes.13,21,22 MFs also contribute to scaffolding and motility of cytoplasmic components as well as provide tracks for the myosin motor proteins involved in vesicle trafficking.10

57.2.3  Membrane Compartments in Epithelial Cells Polarized sorting operations largely occur within the Golgi and endosomes. These membrane organelles have a characteristic subcellular distribution. The Golgi, the chief biosynthetic sorting station, is located in the apical pole in close proximity to the nucleus,13 with tubular structures of the trans-Golgi network (TGN) projecting further toward the apex of the cell. Between the TGN and the apical membrane, an array of small membrane compartments or endosomes constitutes other key sorting stations. These compartments are generally named after their primary function. Apical or basolateral early endosomes (AEEs or BEEs, respectively) are a heterogeneous population of endosomes that localize immediately under their respective membranes, have moderately acid internal pH, and contain the small GTPase Rab5.23,24 Common (recycling) endosomes (CEs or CREs), also called the subapical compartment (SAC) in hepatocytes, are marked by the small GTP-binding protein Rab8.25 In fully differentiated epithelial cells a subapical cluster of membrane tubules is identified by the presence of Rab11 and named apical recycling endosome (ARE).25 Finally, late endosomes or multivesicular bodies (LE/MVB) contain Rab7.26 A detailed description of the specific function of each compartment in protein trafficking will be given in the following section and in Figure 57.2.

57.3  SORTING PATHWAYS Newly synthesized proteins can travel by many different routes to reach their final apical or basolateral membrane destination.27 By maintaining multiple polarized

AEE

ARE

BEE CRE

FIGURE 57.2  Apical and basolateral trafficking routes in epithelial cells.  Known trafficking routes to the apical membrane are shown as solid blue arrows. Lipid raft-associated proteins are thought to transit through the apical early endosome (AEE) and onto the apical membrane. Many non-raft-associated proteins are delivered via the apical recycling endosome (ARE) or common recycling endosome (CRE). Apical proteins may recycle directly from AEE (fast route) or via ARE (dashed blue arrows). Basolateral proteins (red solid arrows) may transit through the CRE or basolateral early endosomes (BEE), and undergo fast recycling via the BEE or slow recycling via the CRE (dashed red arrows). An indirect transcytotic route to the apical membrane (pink arrows) operates via the basolateral membrane and BEE.

trafficking routes, which can overlap with endocytotic sorting and recycling pathways, epithelial cells attain a fast and precise mechanism to physiologically regulate the location and density of cell surface proteins. For the sake of simplicity, we first describe the three main biosynthetic pathways used for trafficking from Golgi to the apical and basolateral membranes followed by a description of the post-endocytic pathways. Both types share multiple compartments, as shown in Figure 57.2.

57.3.1  Direct Delivery Newly synthesized proteins can follow a direct route from the TGN to their final apical or basolateral membrane destination (Figure 57.2). In this scenario, apical and basolateral membrane proteins are sorted soon after their synthesis. 28,29 The TGN has been classically considered to be the main sorting station of the biosynthetic pathway in epithelial cells, but it is becoming evident that sorting events in this route can also take place at various endosomal sorting compartments.30–35 In addition to sorting mechanisms, which segregate apical proteins from basolateral membrane proteins before they reach their final destinations, other mechanisms are involved in the direct delivery pathway. These include directional transport and specific docking of vesicle carriers with the appropriate

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membrane domains. Once delivered to the proper membrane, domain-specific retention may also contribute to maintaining appropriate polarity and cell surface density of specific membrane proteins.

57.3.2  Indirect Pathway Steady-state polarity may also arise by a circuitous routing process, often called the indirect trafficking pathway. In this case, newly synthesized proteins are initially targeted to one plasma membrane domain and then either selectively retained or selectively retrieved and re-sorted to the opposite membrane domain. Such a pathway is favored for many apical membrane proteins in hepatocytes.36 In these cells, the majority of membrane proteins are first targeted from the TGN to the basolateral membrane from where apical proteins are subsequently endocytosed and relocated to the apical membrane. Transcytosis is also present in MDCK cells, where newly synthesized glycosylphosphatidylinositol-anchored proteins (GPI-APs) are delivered first to the basolateral membrane and then, following a transcytotic route, are redirected to the apical membrane.37 Similarly, the polymeric IgA receptor (pIgR) is also endocytosed from the basolateral membrane of MDCK cells and routed to the apical membrane via recognition of a specific transcytosis signal.38–40

57.3.3  Random Pathway Finally, newly synthesized membrane proteins can be randomly targeted to both plasma membrane domains and then be selectively retained or degraded so that polarized sorting is achieved at the plasmalemma. For example, the cystic fibrosis transmembrane conductance regulator (CFTR) and the α2B-adrenergic receptor have been reported to be delivered to both apical and basolateral membranes of MDCK cells, but then they are selectively retained exclusively at the basolateral membrane.41–43 The post-endocytic pathway may also play an important role in the random pathway by selectively recycling specific proteins to the proper polarized membrane. It is important to consider that protein-sorting pathways can be both cell and protein specific. For example, the intestinal brush border protein lactase-phlorizin hydrolase (LPH) is directly delivered to the apical surface of Caco-2 cells,44 whereas the hsuman neurotrophin receptor p75 reaches its final apical destination via a basolateral transcytotic pathway.45 Certain pathways may predominate in a certain cell type, but less traveled routes may be taken, depending on the specific protein. For example, hepatocytes seem to favor the indirect pathway, but canalicular (apical) ATP-binding-cassette (ABC) transporters, multidrug resistance proteins 1 and 2 (MDR1 and MDR2), and sister of p-glycoprotein (SPGS) have been shown to follow a direct biosynthetic delivery pathway.46–48 In some

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cases, the type of epithelial cell defines the route taken by a specific protein. For example, gp80 is sorted to the basolateral membrane of T84 human colonic adenocarcinoma cells,49 but it is expressed on the apical plasma membrane of MDCK cells.50 In other instances, the same protein may be targeted to identical destinations regardless of the epithelial cell type, albeit through completely different trafficking pathways. Such is the case with the human neurotrophin receptor p75, which follows an indirect route to the apical membrane of intestinal Caco-2 cells,45 but is directly routed to the apical membrane in MDCK cells.51

57.3.4  Protein Sorting in the Biosynthetic Pathway 57.3.4.1  Discovery Pioneering studies describing direct apical or basolateral sorting pathways took advantage of the ability of MDCK cells to generate well-polarized monolayers when grown on permeable supports52 and observations that certain viruses emerge from infected MDCK cells in a polarized manner.53 For example, the influenza virus buds exclusively from the apical membrane, whereas the vesicular stomatitis virus (VSV) emerges only from the basolateral membrane. This asymmetric budding response is a consequence of polarized expression of viral coat proteins and has provided an extremely powerful tool to elucidate the mechanisms of asymmetric sorting processes in epithelial cells. Of special interest, the influenza hemagglutinin (HA) coat protein accumulates on the apical membrane, as opposed to the VSV-G coat protein, which localizes exclusively at the basolateral membrane.54 Studies performed using plasmalemma domain-selective detection techniques showed that newly synthesized HA and VSV-G are directly delivered to their target membranes without any transitory stops on opposite plasma membrane domains.28,55,56 A direct trafficking route indicates that proteins must be sorted within an intracellular compartment prior to vectorial delivery to their target membranes. This trafficking pathway was also found to be common for many endogenously expressed proteins, as shown by studies employing a cell surface biotinylation technique57,58 in various cultured cell lines.59–65

57.3.4.2  Golgi is a Key Biosynthetic Sorting Station Immunoelectron microscopic examination of doubly infected MDCK cells showed that both HA and VSV-G glycoproteins traversed the Golgi apparatus and even the same Golgi cisternae.28 Thus, critical segregation steps were thought to take place somewhere between the end of Golgi and the plasma membrane. Support for segregation within the TGN came from observations that traffic

Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

of both apical and basolateral membrane proteins can be arrested at the trans-Golgi by incubation at 19–20°C.66,67 Upon release from the block, viral membrane proteins proceed rapidly and directly to their appropriate apical or basolateral plasma membranes without obligate stops in endosomes or other intracellular compartments.66,68,69 Moreover, following low-temperature Golgi block and release, TGN vesicles containing either apical or basolateral cargo could be separated using equilibrium density gradient centrifugation combined with immunoisolation techniques.70 This provides evidence of a sorting event at the TGN. High-voltage electron microscopy studies combined with computer axial tomography provided evidence for distinct coat-based sorting domains in the TGN, and showed that the TGN consists of multiple tubules that contain clathrin or novel “lace-like” coats.71–73 Additional evidence of apical and basolateral membrane protein sorting at the TGN was obtained using timelapse fluorescence imaging in live cells transfected or infected with basolateral and apical membrane marker proteins. Apical and basolateral membrane cargo progressively segregate within domains of the Golgi and TGN, exclude resident proteins, and then exit in separate tubulovesicular carriers in direct route to the plasmalemma.29,74 Budding and fission of tubulovesicular carriers from the TGN have recently been shown to be controlled by dynamins or dynamin-related large GTPases. Interestingly, dynamin-2 appears to be involved only in fission of tubulovesicular carriers containing the apical marker protein p75, whereas the dynamin-related carboxy-terminal binding protein 3/ brefeldin A-ribosylated substrate (CtBP3/BARS) is specific for tubulovesicular carriers of a the basolateral marker VSV-G.75 Tubulovesicular carriers destined for either apical or basolateral membranes also differ in their composition. A recent study in MDCK cells showed that exit from TGN of many basolateral proteins was impaired by clathrin knockdown, while exit of apical proteins was unaffected.76 Interestingly, trafficking of the Na,K-ATPase β-subunit was also independent from clathrin; this is likely due to the existence of an alternative basolateral membrane sorting mechanism.77

57.3.4.3  Other Sorting Stations for Newly Synthesized Proteins It is becoming increasingly evident that compartments other than the TGN play important roles in polarized sorting. For example, sorting of apical and basolateral heparan sulfate and chondroitin sulfate proteoglycans appears to occur simultaneously with synthesis of their glycosaminoglycan side chains, but prior to their passage through TGN. As a result, glycosaminoglycan chains of glycoproteins destined to the basolateral surface of MDCK cells are shorter and more intensely sulfated than their

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apical counterparts.78–81 In another example, the oligomerization of apical GPI-APs that takes place in rafts or detergent-resistant microdomains (DRMs) within the cis-Golgi is a critical step for their sorting. Paladino et al.82 showed that basolateral GPI-APs were also associated to DRMs in the Golgi, but did not form high molecular weight complexes. As a result, inhibition of raft formation by depleting cells of cholesterol affected sorting of apical but not basolateral GPI-APs.82–84 Polarized trafficking from the TGN to the plasma membrane domains has been shown to follow a direct path, but it should be pointed out that sequential intracellular routing steps involving intermediate endosomal compartments have recently been identified.30,35,85 Orzech et al.30 implemented an assay to measure the meeting of newly synthesized membrane proteins with endosomal compartments loaded with horseradish peroxidase, and found that the biosynthetic road traveled by polymeric immunoglobulin receptors can involve an endosomal compartment — most likely the common recycling endosome.32 Because apical and basolateral proteins intermix in this compartment (see the following section), it is likely that the CRE might also function as a polarized sorting station for some proteins in the biosynthetic pathway.

57.4  POST-ENDOCYTIC PATHWAY Since nearly 50% of a typical polarized plasma membrane is endocytosed per hour,86,87 specific sorting mechanisms must be in place to ensure that endocytosed proteins are recycled back to the appropriate plasma membrane. In addition, sorting in the post-endocytic pathway is also important for polarized distribution of proteins that are processed in the indirect pathway by transcytosis88,89 and for the general maintenance of epithelial cell polarity. Indeed, constant internalization and sorting in the postendocytic pathway may offer a continuous surveillance or proofreading mechanism to relocate proteins that have been randomly sorted or missorted in the biosynthetic pathway. Furthermore, since sorting in the biosynthetic pathway is a one-time event in the lifetime of a protein, continuous postendocytic sorting is quantitatively more important. Study of the intracellular trafficking itineraries of endocytotic recycling (e.g., transferrin and its receptor, TfR) and transcytotic marker proteins (e.g., IgA and its receptor, pIgR) in model epithelia, such as MDCK cells and Caco-2 intestinal cells, has shown at least four distinct populations of endosomal sorting compartments (see Figure 57.2).25,85,90–93 Although more complex in epithelial cells, these endocytic compartments are similar to the early and recycling endosomes that have been well described in nonpolarized cells.87 Internalization of fluid-phase markers from the basolateral or apical surface of MDCK cells revealed that material

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internalized from opposite membrane domains first accumulates in spatially distinct populations of early endosomes (BEE and AEE).94,95 The AEE and BEE appear to be biochemically and physiologically distinct from each other, despite the presence of common biochemical markers, such as Rab 4,96 Rab 5,97 and the Rab effector EEAI.98,99 Both endosomal compartments exhibit different overall protein compositions,100 recycling rates,96 fusigenic properties,100 and dependence on filamentous actin.101 Therefore, it appears that each type of early endosome is designed to support rapid and efficient membrane protein recycling to a specific plasma membrane domain, preventing mixing of apical and basolateral membrane proteins. Material endocytosed into AEE or BEE can be rapidly recycled without passing through intermediated compartments,96 a process dependent on Rab 4.102 In some cases, such as with PDGFβ, recycling through these early endosomes can be subjected to regulation.103 Alternatively, proteins that are not immediately recycled may be targeted into the lysosomal pathway or directed to a common recycling endosome 32,104 (reviewed in 105). The CRE receives and recycles endocytosed material from both apical and basolateral membranes,94,106,107 and it is considered to be a major polarized sorting station (see Figures 57.2 and 57.3). It is generally identified by a perinuclear localization and a tubular morphology, the presence of Rab 1196 and/or Rab 17,108 and absence of fluid-phase markers. Rab 8 has been reported to identify a subset of CRE that is involved in basolateral sorting in kidney cells,109 but regulates apical protein localization in intestinal cells,110 perhaps via an apical-membrane generation complex.111 Because of its clearly differentiated composition, the CRE from MDCK cells can be resolved from

Clathrin

β−arrestin1, epsin1, ARH

DAB2, eps15, epsin1, amphiphysin1

Ear

P

P

P µ2

β2 PIP2

σ2

α

Trunk

Hinge

Clathrin

cargo Yxxφ motifs

early endosomes physically, biochemically, and pharmacologically.96 Ultrastructural studies showed that basolateral sorting of the transferrin receptor takes place in tubular extensions of the CRE112 on clathrin-studded vesicles.113 Similar to the biosynthetic pathway, the sorting process in the CRE is sensitive to Brefeldin A (BFA).113–116 BFA is believed to alter endosomal-sorting processes directly by exerting a well-known inhibitory effect on the association of adaptors and coat proteins (see Sections 57.6.1 and 57.6.1.1) by Arf proteins, which in turn prevents critical segregation steps in the CRE.116,117 Endocytosed apical proteins may also be transferred from AEE to another specialized compartment118 called the apical recycling endosome (ARE),118 which is also called the SAC in hepatocytes. ARE is likely to serve as a depot for membrane proteins traveling between the apical membrane and CRE (reviewed in 119; see Figure 57.2), and it has received a great deal of attention because it is suspected to be a post-endocytic hub where apical membrane traffic is regulated to control apical membrane protein surface density.119 It should be noted that there has been some debate whether ARE is actually a separate endosomal entity or a subdomain of CRE.93 Efforts to differentiate between both compartments have been hampered because of their overlapping or common properties. Both ARE and CRE compartments have a subapical, perinuclear, or pericentriolar cellular location; lack fluid-phase markers; and are dependent on MTs and present a similar set of Rab proteins, including Rab 11, Rab 17, Rab 25, and myosin Vb.93,96,108,120,121 Later studies employing confocal microscopy or studying the segregation of cointernalized marker proteins and fluid-phase markers provided several criteria for distinguishing ARE from the CRE in MDCK

cargo di-Leu motifs

FIGURE 57.3  Schematic representation of the prototypic heterotetrameric clathrin adaptor AP-2.  Binding sites for PIP2 and cargo proteins containing a Yxxϕ internalization motif is found at the β trunk of the subunit, while the di-Leu motif binding site has been shown to be contributed by the α/σ-subunits. Ear domains of both α- and β-subunits act as binding platforms for multiple accessory and regulatory proteins. Regulation via phosphorylation has been proposed to take place at the α-, β-, and μ-subunits.

Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

cells.90,118,122 Brown defined ARE as an endocytic compartment at the extreme apical pole that is enriched in apical proteins. The CRE, in contrast, has an apical and lateral distribution in the cell cytoplasm and contains apical and basolateral proteins in equal proportion. Furthermore, confocal microscopy studies showed that Rab 11 predominately associates with the apical-membrane-proteinenriched vesicles at the extreme apical pole rather than with the CRE. In addition, differences in the intravesicular pH of the CRE (acidic) and ARE (neutral) provide a functional dissimilarity, which can be exploited to differentiate between these two compartments.122 Whether sorting occurs in the TGN or CRE, it generally involves segregation of cargo and the assembly of trafficking vesicles by coat proteins and adaptor molecules or complexes. Selective attachment of a specific coat factor to cargo is believed to promote segregation and concentration of select cargo while specifically marking vesicle carriers for the apical or basolateral membrane. However, selective portioning in specialized lipid domains may provide an alternative mechanism for sorting of apical proteins (see Section 57.3.3).

57.5  SORTING SIGNALS Polarized sorting is directed by signals embedded within the structure of polarized membrane proteins. They are interpreted and acted on by intracellular sorting machineries to segregate, initiate vesicle formation, and transport or retain target proteins on appropriate membrane domains. A great deal of research activity has focused on identifying these signals, usually through mutagenesis and peptide-motif transplantation studies. By definition, polarized expression is lost when a sorting signal is mutated. However, different missorting phenotypes may be observed. In similar cases, proteins can exhibit a complete loss of polarity and become randomly localized to apical and basolateral membranes. In contrast, when dominant signals are deleted and effects of recessive sorting signals are manifested, mutant proteins will be mistargeted to the opposite membrane domain as the wild type. Although mutagenesis studies provide evidence that a structure is necessary for sorting, it is important to demonstrate that the structure is also sufficient for polarized sorting. In this case, the signal should be able to act in an autonomous fashion and confer a polarized sorting phenotype to a reporter protein that otherwise lacks sorting signals.

57.5.1  Basolateral Sorting Signals Sorting of basolateral proteins is typically defined by small amino acid motifs within their cytoplasmic domains. Two different classes of signals have been identified. One class, similar to clathrin-dependent endocytic or lysosomal

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sorting signals (reviewed in 123 ), includes short tyrosinecontaining motifs (NPXY or YXXΦ), where Φ is a hydrophobic residue and is found, for example, in the LDL receptor proximal signal124 or in the VSV-G protein.125 In addition, dileucine/dihydrophobic signals have been identified in, for example, the IgFc receptor,126 the MHC class II protein,127 E-cadherin,128 and NKCC1.129 The similarity of these signals with endocytic motifs, their direct interaction with clathrin-adaptor molecules (see Section 57.6.1), and more recently the role of clathrin in basolateral sorting suggest that both basolateral sorting and endocytosis share some common elements.76 Other basolateral sorting signals do not share any homology to endocytic signaling motifs; they likely represent several distinct kinds of unrelated sorting signals. These include the H/RXXV motif in pIgR, which has been proposed to form a β-turn structure that interacts with BFA-sensitive adaptor complexes to direct basolateral sorting.130 Some bipartite basolateral sorting motifs include the acidic cluster motifs that are either juxtaposed to critical tyrosine residues (e.g., the LDL receptor distal determinant124) or dihydrophobic residues (e.g., in furin131 and the stem cell factor132). Intriguingly, two basolateral sorting signals composed by small acidic clusters followed by proline or hydrophobic residues at variable distances have been recently found in the proton-coupled monocarboxylate transporter MCT3 and MCT4, and promote clathrin-dependent but clathrin-adaptor AP1B-independent basolateral sorting.133 Finally, other basolateral sorting signals are juxtaposed to PDZ binding motifs at the extreme carboxyl-terminus, similar to those in the betaine transporters,134 the Kir 2.3 channel,135 and the GAT-2 GABA transporter,136 although these signals appear to operate independently of the PDZ ligand.

57.5.2  Apical Sorting Signals Apical sorting signals are less well defined, but are traditionally associated with membrane domains and structures that determine clustering, control glycosylation, or dictate membrane microdomains association.137 For instance, specific GPI attachment sequences allow selective partitioning of some apical proteins into lipid rafts at the Golgi apparatus (see Section 57.6.3). In other cases, N- and O-linked glycosylation appears to be required for apical delivery. This may reflect the requirement for proper cargo folding and/or the involvement of apical cargo receptor lectins, such as VIP36, galectin-3, or galectin-4, which may participate in protein sorting or raft-independent clustering events.137 Another example of raft-independent targeting is provided by the gastric H/K ATPase α-subunit, where a transmembrane domain and its flanking regions generate structures that direct this transporter to the apical membrane.138

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Some apical sorting determinants have been recently identified within the protein sequence of cytoplasmic domains of, for example, rhodopsin,139 Na-K-2Cl cotransporter type 2 (NKCC2),129 receptor guanylate cyclases,140 and ATP7B copper-ATPase.141 Interestingly, the endocytic receptor megalin contains apical sorting determinants that include an NPXY-type motif,142 suggesting that apical sorting of some proteins may also involve clathrinadaptor molecules.143

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VAMP7,8 Syntx3+SNAP-23

AP-2 AP-3 AEE

ARE

Rab11

Rab7

57.6  RECOGNITION OF SORTING SIGNALS A great deal of research activity has focused on identifying and characterizing the intracellular machinery that directly interacts with and decodes intracellular sorting signals. Proof that a sorting machinery candidate directly participates in the critical signal recognition event requires evidence that it directly interacts with a specific sorting signal, localizes to the appropriate sorting compartment, and is required for polarized sorting. Disruption of interaction with cargo in vivo should produce the same cargo missorting phenotypes as when the sorting signal is mutated. Although few candidates meet all of these rigorous standards, insights can be gained by a review of our present understanding.

57.6.1  Clathrin Adaptors The similarity of tyrosine- and dileucine-based basolateral sorting signals to those involved in clathrin-dependent endosomal and lysosomal targeting (reviewed in123) long suggested the possibility that basolateral sorting may involve a mechanism similar to clathrin-adaptor-dependent internalization and endosomal trafficking.144 The discovery and characterization of μ1B, an epithelial-specific “medium” subunit of the clathrin adaptor AP-1 complex,145 coupled with observations that clathrin is required for the sorting of many basolateral proteins in MDCK cells,76 strongly suggest that this is the case. Heterotetrameric clathrin-adaptor complexes, called adaptins (AP; reviewed in 146), interact with tyrosine- and dileucine-based motifs (see Figure 57.3). Four adaptor complexes (AP-1 to AP-4) have been described. Each adaptor is composed of two large subunits (γ/β1, AP-1;α/β2, AP-2; δ/β3, AP-3; /β4, AP-4), one medium subunit (μ1–4), and one small subunit (σ1–4).147 Tyrosine-based “YXXΦ” motifs interact with the medium μ1–4 subunits,148,149 within a structurally defined binding pocket.150,151 The smaller σ-subunit and the α-subunit harbor the binding site for dileucine “[D/E]XXXL[L/I]” motifs in AP-2.152,153 Both signal recognition sites are located within the trunk domain of the adaptor complex, far removed from the clathrin binding domains in the hinge region of AP-1 and AP-2. As a consequence, AP-1 and AP-2 adaptor complexes have the

LE

AP-1A

Lys

AP-3

Rab5

?

Rab8, 10 Rab11

EXOCYST

CRE AP-1B AP-2 AP-4?

BEE

Rab5

VAMP3 Syntx4+SNAP-23

FIGURE 57.4  Polarized sorting machinery.  Clathrin-coated vesicles depend on adaptor proteins for many of their intracellular trafficking and endocytic pathways. AP-1A mediates transport from the TGN to the common recycling endosome (CRE). AP-1B plays a role in basolateral trafficking from the CRE. AP-4 mediates non-clathrin-dependent basolateral trafficking, perhaps via endosomes. Proteins may be targeted for degradation from the TGN (AP-1A mediated) via late endosomes (LE), or from apical/basolateral early endosomes (AEE/BEE) via LE, through an AP-3 mediated pathway. Specific Rab GTPases identify different intracellular sorting compartments. Docking of cargo vesicles is mediated by apicalor basolateral-specific SNARE heteromeric complexes made of two target membrane t-SNAREs (typically syntaxin/SNAP-23 proteins) and a vesicle v-SNARE (VAMP).

capacity to simultaneously interact with clathrin and membrane proteins containing tyrosine- or dileucine-based signals. In this way, AP-1 and AP-2 couple cargo recognition to clathrin coat formation.154 AP-3 and AP-4 do not interact with clathrin directly, and it remains unknown if and how these adaptors control vesicle formation. Each AP complex specifies a distinct trafficking operation (see Figure 57.4). The AP-1A complex participates in delivery of cargo from the trans-Golgi to the endosomallysosomal system, AP-1B controls basolateral trafficking from the recycling endosome, AP-2 mediates endocytosis of membrane proteins, AP-3 has been implicated in endosomal and lysosomal delivery,155 and AP-4 has recently been associated with Golgi to endosome transport151 and basolateral traffic.156 Targeting specificity of the different adaptor complexes is governed, at least in part, by their different affinities for different sorting compartments, which is determined by their different phosphoinositide binding properties. Different adaptins also exhibit somewhat different cargo binding properties. The types of YXXΦ and [D/E]XXXL[L/I] signals recognized by each

Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

adaptor complex overlaps to a significant extent, but each AP complex exhibits differing preferences for residues neighboring the critical tyrosine residue151,157 and the dileucine motif.158

57.6.1.1  AP-1B Sorting of many, but not all, basolateral proteins require the AP-1B complex (γ/σ1/β1/μ1B). Distinguished by the presence of its epithelial-specific μ1B-subunit, the AP-1B is biochemically and spatially distinct from the ubiquitous AP-1A adaptor (γ/σ1/β1/μ1A).159 It localizes to the CRE rather than the TGN,159 where it performs sorting functions for proteins traveling to the basolateral membrane in the biosynthetic and post-endocytic pathways.35,160 This discovery evolved from elegant studies that made use of unusual properties of a pig kidney epithelial cell line, LLC-PK1.161 LLC-PK1 cells lack AP-1B and “mistarget” proteins containing tyrosine-based basolateral sorting signals to the apical membrane.145,162 When μ1B-subunits are stably expressed in LLC-PK1cells, however, they co-assemble into heterotetrameric AP-1B complexes and allow basolateral sorting of target proteins that are otherwise apically expressed in wild-type LLC-PK1 cells.162 The most likely explanation for the requirement of AP-1B is that it directly interacts with tyrosine-based sorting signals, recruiting proteins containing them into clathrin-coated vesicles (CCVs) for the transport to the basolateral membrane. Interestingly, many of the signals recognized by AP-1B, such as the one in the LDLR, do not conform to the canonical YXXΦ adaptin-binding motif.124 Other proteins, such as the TfR, require μ1B for their sorting but rely on sorting signals that share no resemblance to any known clathrin-adaptor binding motif (see Section 57.5.1). Some of these signals may interact with μ1B through a binding site other than that used by YXXΦ motifs. In support of this idea, mutant μ1B-subunits rendered defective for YXXΦ binding are unable to support basolateral membrane targeting of proteins containing such a motif, but they are still able to support basolateral membrane expression of LDLR and TfR.163 It should be emphasized that there are alternative routes to the basolateral membrane that are completely independent of AP-1B or even clathrin.76,77 For example, the IgG Fc receptor FcR11-B2,161 the Kir 2.3 channel,135 or the Na,K-ATPase77 depend on an alternative but still uncharacterized basolateral sorting mechanism. The FcR11-B2 contains a dileucine-type basolateral targeting signal,161 which presumably has the capacity to interact with α-adaptin-subunits. Thus, other adaptin complexes (such as AP-4; see the following section) or adaptin-like molecules may also be involved in the basolateral targeting of some of these proteins.

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57.6.1.2  AP-4 The adaptor protein AP-4164,165 is expressed in both polarized and non-polarized cells, where it localizes to the TGN and endosomes.147,156,164,165 It has been found to interact in vitro with the basolateral sorting signals of furin,131 LDLR,114 and the TfR.166 Importantly, RNA antisensemediated studies revealed that the μ4-subunit in MDCK cells is required for basolateral sorting of LDLR and furin, but not transferrin.156 Thus it is a likely candidate to mediate recruitment of cargo for basolateral trafficking in a clathrin-independent and AP-1B-independent sorting pathway (see Figure 57.4). Recent studies with the AP-4 routing-dependent Alzheimer’s disease-related amyloid precursor protein (APP)150,151 raise the possibility that AP-4 might mediate basolateral delivery through a TGNto-endosome routing pathway.

57.6.2  Vesicle Formation and Budding Sorting signal recognition is often coupled to vesicle formation. Coated vesicles have a critical role in concentrating, packaging, and shuttling cargo between different intracellular compartments and plasma membrane domains (reviewed in 167,168). Three different types of coated vesicles have been described based on their compositions and the intracellular compartments where they originate. CCVs mediate transport between TGN, endosomes, and the plasma membrane. Coatomer complex I (COPI)- and II (COPII)-coated vesicles mediate intra-Golgi or Golgi to endoplasmic reticulum (ER) retrograde transport and ER to Golgi transport, respectively. Formation of all three types of coated vesicles shares a common sequential mechanism in which coat subunits assemble on the membrane and recognize cargo. As cargo and coat proteins concentrate, the underlying membrane deforms, buds from the parent membrane, and is cut off, forming a coated vesicle. The three types of coated vesicles recognize specific sets of cargo and have different protein and lipid compositions. CCVs are composed of two cytosolic protein complexes, clathrin triskelia and heterotetrameric adaptor complexes (AP-1 and AP-2). COPII vesicles have a similar structure, but the two protein complexes are Sec13–31 and Sec23–24. The COPI coat is somewhat more simple and contains a single multi-subunit coatomer complex. Despite their differences, all of these protein complexes have a common tendency to self-assemble into empty spherical cages,169–171 similar to the outer cage found in vesicles in vivo. Cage subunits cannot bind directly to the membrane, and require an inner adaptor layer capable of simultaneous interaction with cage protein complexes, sorting motifs in cargo proteins, and the compartment membrane via negatively charged lipids.168,172 Sculpting the membrane into the curved shape of a vesicle is an energy-intensive process that requires

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recruitment of additional proteins. Two related GTPases, Arf1 in COPI vesicles and Sar1 in COPII vesicles, appear to manage both coat recruitment and curvature generation.167 A similar role is played by the GTPase dynamin in CCV, although additional proteins such as amphiphysin, epsin, endophilin, and members of the sorting nexin family are also recruited and play a role in binding to members of the core component, or recognizing, generating, and stabilizing the membrane curvature.167,173 GDP-bound Arf1 associates with the membrane, and upon GTP binding it undergoes a conformational change. This change exposes a short amphipathic N-terminal helix that is inserted into the lipid bilayer, causing an asymmetric expansion of the outer versus the inner leaflet, allowing Arf1 to remodel the membrane into highly curved structures.174,175 Similar to Arf1, Sar1 action also follows a cycle of GTP binding followed by association to the membrane, recruitment of the adaptor complex Sec23–24, and a conformational change that induces curvature of the membrane.176–178 Formation of CCVs appears to be more complex, with multiple accessory proteins interacting with the membrane through structural elements such as Bin, amphiphysin, Rvs (BAR) or epsin N-terminal homology (ENTH) domains.179 In addition, dynamin contains a PH domain that provides docking sites for negatively charged lipid headgroups. GTP-bound dynamin readily associates with the membrane and inserts its PH domain in the lipid bilayer, inducing curvature of the membrane. The scaffolding role of dynamin allows this protein to interact with multiple SH3-domain containing accessory proteins, which in turn contribute to stabilize the membrane curvature.180,181 Finally, dynamin has also been shown to play an important role in vesicle scission and release from the donor membrane, a process also known to require GTP hydrolysis.178

57.6.3  Apical Routing Via Lipid Rafts and Associated Molecules Sorting signals may also govern lipid partitioning into membrane domains like “rafts.” In fact, lipid rafts were first defined as membrane microdomains rich in glycosphingolipids and cholesterol, which serve as sorting platforms and vectorial delivery vehicles for apical membrane proteins and lipids traveling in the biosynthetic pathway.182 In supporting this model for apical trafficking, nearly all endogenous GPI-APs expressed in MDCK were apically localized64,183 due to their incorporation into DRM domains as they reach the Golgi complex.184 In addition, depletion of glycosphingolipids or cholesterol results in missorting of apical GPI-APs.185 The correlation between GPI-linkage, DRM association, and apical membrane delivery suggested that GPI anchors might serve as raft-dependent apical sorting signals. This view was

SECTION  |  V  Digestion and Absorption

initially reinforced by the observation that chimeric GPIAPs are directed to the apical membrane.65,186 The results of these studies were challenged by the observation that in Fisher rat thyroid cells the majority of endogenous GPI-APs are delivered to the basolateral membrane,183 whereas in hepatocytes they are first targeted to the basolateral membrane and then transcytosed to the apical domain.187,188 What is more, in MDCK cells not all lipid raft-associated proteins are apical189,190 or affected by cholesterol depletion.191,192 Furthermore, in MDCK cells some GPI-APs are also delivered first to the basolateral membrane and then move to the apical surface by transcytosis.37 The controversy of whether glycosylphosphatidylinositol anchors (GPI anchors) are true apical sorting signals is beginning to be clarified by recent studies showing that not all GPI anchors are the same, and by the realization that raft association is a more complex process than originally thought. Both apical and basolateral GPI-APs associate with DRMs, but only apical GPI-APs cluster into large oligomers that are selectively routed to the apical membrane.82,193 A mechanistic understanding of this critical clustering event has been provided in a recent work by Kinoshita et al.194. This shows that the unsaturated fattyacid chain present in GPI anchors at the ER undergoes a considerable remodeling as it transverses the Golgi, and is turned into a saturated fatty-acid chain by specialized Golgi-resident enzymes.194,195 This fatty-acid maturation is critical for the association of GPI-APs into lipid rafts. Variations in the sensitivity of individual GPI anchors to these enzymes condition their final inclusion into lipid rafts137,189,196 and, consequently, determine whether they are targeted to the apical or basolateral membrane. GPI anchors are the most common raft association signal, but an alternative way of transmembrane-domaindependent raft association has been described for HA and neuraminidase, which contain apical sorting determinants within their transmembrane domains.197,198 Disruption of DRM by cholesterol removal causes missorting of these proteins to the basolateral membrane,185,199 and DRM association is governed, at least in part, by their transmembrane apical sorting signals. Association with raft-associated molecules also plays a role in targeting proteins for inclusion in lipid rafts. For example, in intestinal cells, galectin-4 participates in lipid raft clustering via interaction with glycosphingolipids and N-linked complex carbohydrates in raft-associated glycoproteins.200,201 Annexin II and annexin 13b are Ca2dependent lipid-binding proteins that associate with DRM and are required for raft-dependent trafficking of some apical membrane proteins.202–204 Annexin 13b directly interacts with an apical-membrane-directed MT motor protein, KIFC3, suggesting a role in apical-membrane-directed delivery of DRMs.205 Annexin II plays a role in the sorting

Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

and transport of brush border hydrolases, such as sucroseisomaltase, as well as structural components like ezrin, and may even play a broader role in maintaining intestinal cell polarity. Downregulation of annexin II in Caco-2-A4 cells results in a severe reduction of the levels of the brush border membrane resident enzyme.206 It should be pointed out that lipid microdomain association can play a role in basolateral polarity. Some basolateral membrane proteins also partition into specialized cholesterol-enriched compartments207,208 called caveolae, which are primarily found on the basolateral membrane in intestinal cells,209 native kidney cells,210 and MDCK cells.211,212

57.6.4  The Rab GTPases Rab GTPases regulate the spatial and temporal organization of numerous membrane trafficking processes, including vesicle budding, targeting, and tethering as well as the docking and priming stages of vesicle fusion. The precise details of Rab function are still hazy, but Rab proteins appear to orchestrate trafficking events by specifically associating with a precise membrane compartment that contains a unique set of effector proteins (reviewed in 213), making them ideal components of polarized sorting machines. Several Rabs have been implicated in apical membrane sorting processes (see Figure 57.4). The epithelialspecific Rabs, Rab 17214 and Rab 25,215 along with the more widely expressed Rab 11a, localize to apical endosomes.120,216 In gastric parietal cells Rab 11a, along with Rab 25 and the SNARE proteins syntaxin 3 and VAMP2 (see Section 57.8.1), appear to be critical in promoting regulated apical membrane insertion of the H,K-ATPase responsible for acid secretion into the secretory canalicular membrane.216 Rab 17 has been implicated in the regulation of traffic through ARE in MDCK.108 More recently, Rab 14 has been found to localize to the TGN and apical endosomes to regulate delivery of cargo to the apical domain.217 Other Rabs, particularly Rab 8, have been implicated in basolateral membrane processing steps for proteins traveling in the μ1B adaptor-dependent pathway in MDCK cells,109,218 although surprisingly, the same Rab has been found to regulate apical membrane localization in intestinal cells.110 In addition, Rab 10 has been proposed to participate in basolateral targeting form CRE.219 Interestingly, Rab 3b has been found to interact with a specific 14 amino acid site in the cytoplasmic domain of the pIgR receptor that was previously shown to be required for transcytosis.220 Regulated dissociation of Rab 3b from pIgR stimulates basolateral to apical pIgR transcytosis, presumably by triggering translocation of receptor-containing vesicles from the ARE to the apical membrane. In this way direct interaction of vesicle cargo with a Rab can

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determine its own intracellular traffic. A direct cargo interaction mechanism may have evolved for very abundant membrane proteins, like pIgR, to provide a dedicated high fidelity and robust trafficking mechanism.

57.7  POLARIZED TRANSPORT AND DELIVERY For polarized protein expression to be achieved, transport carriers must be correctly delivered to the appropriate acceptor membrane. Current evidence indicates that longrange transport may be carried out by MTs, whereas shortrange transport near the cell surface is carried out by actin. In addition, specific sets of molecular motors appear to be involved in apical and basolateral trafficking.10

57.7.1  Microtubule-mediated Transport The unique organization of MTs in polarized epithelial cells (see Section 57.2.1.1), along with the presence of directional MT-motor proteins, has long suggested an important role for MT in apical and basolateral membrane transport. MTs have been shown to facilitate the rapid (~0.1–1 μm/sec) and efficient movement of vesicles to the plasma-lemma (reviewed in221). Apical traffic is generally more sensitive to MT disruption with agents such as nocodazole or colchicine than basolateral traffic. MT disruption affects delivery of proteins to the apical membrane whether they are traveling in the biosynthetic,222,223 the apical recycling,224 or the transcytotic pathways.225 Basolateral trafficking is more resistant to MT disruption, and some proteins are more sensitive than others.222,226,227 More recently, similar conclusions have been reached using real-time imaging techniques29 to directly monitor the role of MT in trafficking and fusion of post-Golgi transport intermediates carrying apical or basolateral-destined proteins. MT motors are remarkably selective, associating with select vesicles to ensure movement in the appropriate direction. In some cases, MT motors may associate with a transport vesicle by directly interacting with the cargo proteins. For example, the dynein light chain directly interacts with an apical sorting signal in the COOH-terminal tail of rhodopsin to mediate trafficking from TGN to the apical membrane.139,228 Sometimes, transport of one cargo may require sequential intervention of two different motor proteins at different steps. For example, the apical marker p75 requires dynein for release of vesicles from the TGN and kinesin for the formation and transport of apically directed tubulovesicular carriers.229 Minus-end-directed motors, like dynein and the unconventional kinesins, selectively carry vesicles toward the apical membrane. Conventional kinesin motor proteins are plus-end-directed and transport vesicles to the basolateral membrane, as well as along the mixed polarity subapical

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MT network. For example, in the renal collecting duct, dynein participates in transport of AQP2 water channels to the apical membrane.230 Trafficking of the human sodiumdependent multivitamin transporter (hSMVT) in intestinal cells appears to involve distinct trafficking vesicles that require an intact MT network and the motor protein dynein for their mobility.231 KIFC3, a minus-end-directed kinesin, transports HA and annexin XIIIb to the apical membrane.205 In contrast, the plus-end MT molecular motor kinesin-1 participates in traffic of the Na,K-ATPase to the basolateral membrane of alveolar cells. Intriguingly, conventional plus-end-directed kinesin motors (KIF5 family) have been shown to also support traffic of vesicles to the apical membrane by using a recently discovered highly dynamic population of MTs that preferentially grow their plus-ends toward the apical pole.16,137 For example, KIF5B supports the TGN exit of the apical marker p75,16 and KIF5C mediates a similar role for both the raft-associated sucrose-isomaltase and the raftindependent neurotrophin receptor.232

57.7.2  Actin-mediated Transport Transport along actin filaments, slower than transport along MTs, is driven by myosin motor proteins which, except for myosin VI,233 move toward the barbed () end of actin filaments.234 Recent studies on myosin motors have provided new insights into the role of the actin cytoskeleton in polarized trafficking. For example, myosin Vb interacts with Rab 11 in CRE and is involved in traffic between the ARE and the apical surface.216,235,236 It has been suggested that myosin I plays a role in raft-associated apical traffic of sucrase-isomaltase in intestinal cells.237 Myosin motors have also been shown to play significant roles in basolateral traffic. For example, myosin IIa is found at the Golgi apparatus and has been shown to promote selective exit of basolateral proteins.238 Myosin VI has been shown to be required for sorting of basolateral cargo in the m1B-dependent pathway in MDCK cells.239 In addition, Myosin-Vc is expressed primarily in epithelial

(A)

cells and interacts with Rab 8, suggesting a possible similar role in a basolateral m1B-dependent sorting pathway.240

57.8  POLARIZED DOCKING AND FUSION Specific molecular machinery provides the force required for fusion of donor and acceptor membranes, exerting a critical control to ensure that an individual vesicle fuses with the right target membrane, providing a final “proofreading” step in the polarized protein trafficking process.

57.8.1  v-SNAREs and t-SNAREs The core machinery for vesicle fusion is comprised of a family of structurally related integral membrane proteins known as soluble N-ethyl maleimide-sensitive attachment factor receptors (SNAREs), and two soluble proteins named N-ethyl maleimide-sensitive factor (NSF) and soluble NSF attachment factor (α-SNAP). The initial step in this membrane fusion event involves the interaction in cis of two SNAREs at the target membrane (t-SNAREs), which are generally homologs of the syntaxin and/or SNAP-25. The resulting heterodimeric complex then interacts in trans with a vesicle SNARE (v-SNARE), which is generally a VAMP/synaptobrevin homolog, to form a fusion complex (see Figure 57.5).241 The original SNARE hypothesis242 proposed that fusion selectivity was achieved by the specificity of pairing between v-SNAREs and t-SNAREs. Thus, restriction of each SNARE to a specific donor and acceptor membrane would warrant precise docking in a given pathway. Early studies showed that basolateral trafficking was affected by clostridial neurotoxin-mediated VAMP proteolysis, whereas apical traffic was toxin-insensitive, suggesting a SNAREindependent mechanism for apical sorting. Later studies showed that a toxin-insensitive VAMP-7 mediates TGNto-apical membrane transport.243 Furthermore, the equally toxin-insensitive VAMP-8/endobrevin cycles between the apical membrane and endosomes.244 Additional SNARES may also be associated with polarized sorting in epithelial

(B)

FIGURE 57.5  Schematic diagram of the SNARE-catalyzed membrane fusion process.  (A) The t-SNARE is composed of the integral membrane protein syntaxin (blue) and the SNAP-25 soluble protein (green) anchored at the target membrane via a palmitoyl side chain. The t-SNARE interacts in trans with the v-SNARE (a VAMP/synaptobrevin, red) at the vesicle membrane to form a trans-SNARE or SNAREpin complex, which generates an inward force that induces both membranes to fuse. (B) After fusion, the SNAREs remain attached to the resulting membrane as a low energy cisSNARE complex.

Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

cells (see Figure 57.4). In native intestine and many other polarized epithelial cell types syntaxin-3 localizes to the apical membrane, whereas syntaxin-4 is found exclusively at the basolateral membrane.245,246 In contrast, the t-SNARE SNAP-23 associates with both syntaxins in their respective membrane domains.247 Recently, syntaxin-1A has been shown to regulate exocytotic insertion of HATPase-containing vesicles and also in complex with SNAP-23.248 Some v-SNARESs have also been implicated in polarized membrane traffic. In renal cortical collecting, duct principal cells, VAMP2 and VAMP3, along with SNAP23, are present in subapical intracellular vesicles that contain the AQP-2 water channel. Protein knockdown of VAMP2, VAMP3, and SNAP23 was shown to reduce levels of AQP2 fusion at the apical membrane.249,250 Likewise, VAMP2 associates with SNAP-23 in subapical vesicles containing the H-ATPase in intercalated cells.251 VAMP-3 is also required for basolateral sorting of the AP-1B-dependent cargo transferrin and truncated LDLR in MDCK cells.252 Whereas the polarized distribution of some SNAREs in epithelia is apparent, SNARE pairing promiscuity, among other factors, suggests that fusion specificity might be encoded by factors other than t-SNARE/v-SNAREs partnerships.253 For example, the epithelial-specific protein Munc-18-2 interacts with syntaxin-3 at the apical membrane of intestinal epithelial cells and specifically inhibits trafficking of apical cargo.254–257 Other factors, such as membrane tethers and Rab proteins, appear to coordinate initial docking events preceding SNARE-mediated fusion.258 The exocyst serves as a prototype.

57.8.2  Role of the Exocyst in Basolateral Trafficking The exocyst is a multiprotein complex of eight proteins (sec3p, sec5p, sec6p, sec8p, sec10p, sec15p, Exo70p, and Exo84p) first identified in budding yeast259 that functions as an exocytic machinery and mediates polarized tethering and/or docking of post-Golgi vesicles at the growing bud tip. In mammalian epithelia, the evolutionarily conserved complex260,261 appears to operate as a tethering and insertion mechanism for some vesicles destined for the basolateral membrane.261–264 The exocyst in the yeast bud tip provides insights into how it might function at the basolateral membrane. In yeast, assembly of the exocyst complex is controlled by the vesicle-associated Rab GTPase Sec4, whereas docking is specified by Rho-dependent localization of Sec3.265 The active, GTP-bound form of Sec4 directly interacts with Sec15, triggering assembly of the entire exocyst complex. As a result, Sec4p on the secretory vesicle surface becomes indirectly tethered to Sec3 on the cell surface.266 Intriguingly, the most likely mammalian

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ortholog of Sec4, Rab 8, has been strongly implicated in basolateral membrane trafficking,109,218 suggesting a parallel between yeast and mammalian epithelia. In contrast to the yeast exocyst, the mammalian exocyst in epithelial cells Sec3p is neither a Rho effector nor does it act as a spatial landmark for exocytosis on the lateral membrane.261 In MDCK cells, cytosolic Sec6 and Sec8 are rapidly recruited to sites of cell–cell contact upon initiation of calcium-dependent cell–cell adhesion. They become localized to a zone near the junctional complex on the lateral membrane, but the underlying mechanism is not well defined. Additional binding partners have been identified in mammalian systems. The mammalian exocyst is a target of the Ral GTPase. Direct interaction of Ral GTPases with the exocyst components Sec5p267,268 and Exo 84p264 facilitates assembly of the entire exocyst complex.264,267 Perturbation of Ral signaling causes inhibition of exocyst assembly and missorting of several basolateral membrane proteins to the apical surface of polarized epithelial cells,264,267 demonstrating the important role of the exocyst in polarized targeting of basolateral membrane-proteins.

57.9  SELECTIVE RETENTION Selective retention mechanisms play critical roles in the polarized distribution of proteins beyond its important function in the random sorting pathway. Indeed, the surface density of many different types of proteins is tightly controlled at their polarized locales by a dynamic balance between retention and internalization. The best characterized retention factors are PDZ-domain containing proteins (Table 57.1).269 PDZ domains, named after the homologous group of proteins from which they were originally identified, PSD 95 (post synaptic density protein),270 Dlg (Drosophila Disc large tumor suppressor), and ZO-1 (zona occludes271), are ~100 amino acid protein-interaction modules that bind short protein motifs (four amino acid). These are generally,272 but not always,273,274 found at the extreme COOH-terminus of target proteins. PDZ proteins usually have multiple protein–protein interaction modules, allowing them to act as scaffolds that bring proteins together into macro molecular complexes. Members of the NHERF family of apical PDZ proteins provide an excellent example. The scaffolding functions of NHERF1 and NHERF2 are made possible by their two PDZ-binding domains and an ezrin/radixin/moesin/(ERM)binding domain.275,276 NHERF interacts with Na/H exchanger isoform 3 (NHE3), the transporter responsible for the majority of neutral sodium absorption in the intestine and renal proximal tubule, through its PDZ domains, while it simultaneously engages the actin cytoskeleton through ERM protein binding. As a result, NHE3 can be effectively retained in the apical microvilli.277 These interactions are highly regulated to control the location

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TABLE 57.1  PDZ Adaptors, Interacting Proteins, and Function in Gastrointestinal Epithelia PDZ adaptor

Function

Location

Binding Protein and References

NHERF1

Apical scaffolding

Small intestine

NHE3,283 CFTR, β2-AR,284 SLC26A3285

Liver (hepatocytes)

MRP2286

NHERF2

Apical scaffolding

Small intestine

CFTR, LPA2R,284 SLC26A3285

PDZK1 (NHERF3)

Apical scaffolding

Small intestine

OCTN2, PEPT1,287 OATP1A,288 NHE3,285 CFTR,284 SLC26A6289

Liver (hepatocytes)

OATP1A1,290 SR-BI291

PDZK2 (HERF4 or IKEPP)

Recycling in subapical compartments

Small intestine

NHE3292

Shank2 (CortBP1)

Apical scaffolding

Pancreatic ducts, hepatocytes, colon

CFTR, NHE3293–295

Lin-7/CASK

Basolateral scaffolding

Small intestine

Unknown296

SAP97

Basolateral, cell–cell adhesion

Ubiquitous

Unknown297

GOPC (PIST, FIG or CAL)

Golgi/TGN. Modulation of protein expression

Small intestine

CFTR298

of NHE3 in the apical microvilli. In the proximal tubule, for example, binding ties between NHERF1 and NHE3 become broken upon exposure to PTH, dopamine, or an acute rise in blood pressure, causing a myosin VI-driven translocation process to move NHE3 toward the base of the microvilli into raft structures where transport activity is turned off.278,279 NHERF also regulates NHE3 by mutual retention and recruitment of signaling molecules and other transporters. In mouse jejunum, for example, NHERFdependent retention and scaffolding provides a mechanism to coregulate NHE3 and the Na-glucose cotransporter, SGLT1, which is important for oral rehydration when diarrhea occurs.280 A balance between membrane protein retention, internalization, and recycling determines the steady-state localization of the target protein. In fact, target proteins may have different fates following retention-protein detachment, depending on how they are internalized and recycled. Such is the case with proteins that interact with the basolateral membrane PDZ protein complex, which are comprised of Lineage protein 7 (Lin-7) and the calcium-/ calmodulin-dependent serine protein kinase (CASK). Although this complex retains many different proteins at the basolateral membrane, these proteins exhibit different trafficking phenotypes when they become disengaged from Lin-7/CASK.25 Some proteins, like the Kir2.3 potassium

channel, are redirected to an endosomal compartment, consistent with strong endosomal targeting signals, whereas others, like the betaine-GABA transporter (BGT1), maintain basolateral localization, presumably because of the rapid recycling rates.281 In contrast, when chimeric LET23/nerve growth factor receptor protein loses its ability to engage Lin7/CASK it redistributes to the apical membrane.282 In this case, basolateral retention limits transcytosis of these receptors to the apical membrane.

57.10  SUMMARY The asymmetric distribution of proteins on apical and basolateral surface membranes makes the functions of epithelial cells in the GI tract possible. Highly coordinated protein segregation, sorting, recycling, and retention processes are the chief underpinnings of epithelial polarity. Polarized targeting is initiated by sorting signals in membrane proteins, which mark them as cargo for inclusion in vesicles that are destined for a specific polarized cell surface. Associated motor proteins, SNAREs, and tethering complexes control polarized vesicle trafficking and fusion. Other signals in surface proteins control interaction with polarized retention complexes. In recent years, much has been learned about mechanisms of vesicle trafficking fusion and polarized retention, but the

Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

intracellular trafficking machineries that decode and act on polarized sorting signals still remain poorly understood. New advances in this area should provide new insights into physiology and pathophysiology of the GI tract.

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SECTION  |  V  Digestion and Absorption

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Chapter  |  57  Molecular Mechanisms of Protein Sorting in Polarized Epithelial Cells

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