TRENDS in Cell Biology Vol.11 No.12 December 2001
Protein trafficking in the exocytic pathway of polarized epithelial cells W. James Nelson and Charles Yeaman
Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.
Polarized epithelia form permeability barriers between two compartments in the body and vectorially transport ions and solutes between these compartments to maintain ionic homeostasis (Fig. 1). Long before cell biologists became interested in mechanisms involved in protein sorting in epithelial cells, physiologists recognized that vectorial ion transport required the separation of ion channels and transporters into two structurally and functionally different plasma membrane domains, termed apical and basal-lateral, that faced the compartments separated by the epithelium1 (Fig. 1). In the 1980s, cell biologists began to analyze protein sorting and trafficking in cell culture models of simple polarized epithelia from intestine (Caco-2) and kidney (MDCK, LLC-PK1). Although sorting of membrane proteins was described, the mechanisms involved were unknown. However, during the period 1991–2001 – the focus of this review – investigators identified mechanisms involved in protein sorting in the trans-Golgi network (TGN) and plasma membrane. Sorting and transport of proteins to apical and basallateral membrane domains
W. James Nelson* Charles Yeaman§ Dept of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA 94305-5435, USA. *e-mail: [email protected]
§Present address: Dept of Anatomy & Cell Biology, University of Iowa, Iowa City, IA 52242, USA.
The legacy of studies from the 1980s was a general belief that newly synthesized proteins were sorted in the Golgi complex and delivered to either the apical or basal-lateral membrane (Fig. 1); a complication, however, was a study in hepatocytes in situ that showed clearly that the site of sorting was the plasma membrane2. The sorting mechanisms and basis for different sorting locations were unknown. It was suggested that apical sorting might be mediated by lipid sorting in the TGN, a mechanism specific to polarized epithelial cells as apical proteins were generally found only in polarized epithelial cells3. On the other hand, basal-lateral proteins appeared to be constitutive to all cell types and hence might be sorted by a default mechanism. http://tcb.trends.com
During the 1990s, careful mutagenesis of protein sequences and analysis of mutant protein trafficking in polarized cells defined sorting signals in proteins. Given the emphasis on sorting of apical membrane proteins at that time, it was unexpected that the first sorting motif identified was one for basal-lateral sorting and was based on the mutagenesis of hemagglutinin, which normally is sorted to the apical membrane4,5. Other studies examined in detail minimum sequences to sort bone fide basal-lateral membrane proteins, such as the pIgA-receptor6 and the low-density lipoprotein (LDL) receptor7,8. Importantly, it demonstrated that a short sequence was sufficient to redirect a heterologous reporter to the basal-lateral surface9. Together, these types of study provided the first solid information about sorting motifs in basal-lateral membrane proteins. Basal-lateral sorting signals are located in the cytoplasmic domain (Fig. 2) and frequently contain a crucial tyrosine residue within the amino acid sequence NPXY or YXXφ (where X is any amino acid and φ is an amino acid with a bulky hydrophobic group). In several cases, these motifs are predicted to form a structure known as a tight β-turn6,8. In some cases, a dihydrophobic motif serves to target the protein basallaterally10. However, a growing number of basal-lateral sorting signals bear little or no sequence similarity to these motifs, although some of these latter signals might form a peptide structure that comprises a tight β-turn and therefore resemble the tyrosine-based signals6,11. It is unknown whether these structures are present in the context of the native protein. How do these motifs specify protein sorting to the basal-lateral membrane? The YXXφ- and dihydrophobic-based sorting signals selectively bind to clathrin adaptor protein complexes AP-1 and AP-2, and tyrosine-based signals specifically bind to the medium (µ) chains of these complexes12 (Fig. 2). Recent evidence indicates that di-hydrophobic signals bind to the β-chains, rather than the µ chains of adaptor complexes13. Although interactions between tyrosineand di-hydrophobic-based signals and AP-1/AP-2 complexes are responsible for cargo-selective sorting into TGN- and plasma membrane-derived clathrincoated vesicles, the mechanism responsible for basallateral sorting was unknown until very recently. Expression of a recently discovered medium chain for AP-1B, µ1B, located on TGN membranes, correlates with correct sorting of LDL-receptor to the basal-lateral membrane domain, and a complex of LDL-receptor and AP-1B could be isolated14,15. These results indicate
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Fig. 1. Schematic representation (a) of a simple, transporting epithelium (for example, renal tubule) comprising a closed monolayer of cells surrounding a fluid-filled lumen; transport of ions and solutes is from the lumen of the tubule across the epithelium to the serosa (red arrow). Each epithelial cell (center) in the monolayer comprises an apical (green) and basal-lateral membrane (black) that are separated by a tight junction (purple sphere). Studies in the 1990s revealed vesicle trafficking/sorting pathways (b) and an unusual organization of the cytoskeleton (c) in polarized epithelial cells.
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(a) Simple (transporting) epithelium (b) Vesicle trafficking pathways
(c) Cytoskeleton organization Apical membrane
Apical sorting pathway
Basal–lateral sorting pathway Golgi complex
Microtubules N +
+ Basal–lateral membrane TRENDS in Cell Biology
that the AP-1B adaptor complex is involved directly in sorting a class of basal-lateral membrane proteins in polarized epithelial cells. In the future, it will be important to determine how many other proteins are sorted by the AP-1B complex. Another recently described adaptor complex, AP-3, also interacts with tyrosine- and di-hydrophobic-based signals16 and might be involved in the formation of a novel class of clathrin-coated vesicles in the TGN and endosomes. The overall concept of how protein sorting occurs in the TGN changed with these results. That an apical membrane protein could be converted to a protein sorted to the basal-lateral membrane demonstrated that both apical and basal-lateral proteins were actively sorted from each other in the TGN, rather than one pathway being specific (apical) and the other default (basal-lateral). That the basal-lateral pathway could be saturated, with the result that ‘excess’ protein was delivered to the apical membrane, indicated that proteins have multiple signals for apical and basallateral sorting, and recognition of those signals in the TGN appears to be hierarchical, with basal-lateral sorting dominant over apical sorting17. In contrast to the importance of the cytoplasmic domain in protein sorting to the basal-lateral membrane, protein sorting to the apical plasma membrane appears to be dependent on motifs present in the lumenal domain or membrane-anchor18. Strong evidence supports a role for a glycosylphosphatidylinositol anchor in apical sorting in some cells19–21, although this signal does not function as an apical-sorting determinant in all epithelial cells22 (Fig. 2). Apical sorting has been proposed to involve clustering of proteins into glycolipid- and cholesterolcontaining membrane domains, or ‘rafts’, in the exoplasmic leaflet of the Golgi23 (Fig. 2). Production of apical transport vesicles has been suggested to involve VIP21/caveolin24,25, a cholesterol-binding protein that is enriched in ‘rafts’in the Golgi. However, apical transport of many other proteins occurs independently of rafts26. Therefore, apical sorting is likely to involve more than one mechanism, but the details of these mechanisms are unclear. It has been suggested that N-glycans play a role27; however, apical sorting of many proteins occurs independently of N-glycosylation28 (Fig. 2). Mechanisms of http://tcb.trends.com
carbohydrate-mediated apical sorting are not known. One potential mechanism involves recognition of carbohydrate moieties by sorting lectins, such as VIP36 (Ref. 29), which is hypothesized to partition into microdomains of the TGN along with other apical membrane proteins and lipids. Apical and basal-lateral membrane proteins that have been sorted into distinct subdomains of the TGN must be packaged into transport vesicles. High-voltage electron-microscopy studies have revealed that the TGN comprises multiple distinct membrane tubules that possess either clathrin coats or a novel ‘lace-like’ coat30. Each tubule produces only one type of vesicle, which suggests that protein sorting and vesicle budding are distinct. However, vesicle budding mechanisms are not understood. One protein implicated in the budding of basal-lateral transport vesicles is p200/myosin II (Ref. 31; but see also Ref. 32). In MDCK cells, p200 is present on basal-lateral transport vesicles and absent from apical transport vesicles31. Recent studies indicate a role for the small GTPase cdc42 in the formation of vesicles from the TGN (Ref. 33) and subsequent delivery to the plasma membrane34, suggesting a role for the actin cytoskeleton; but the mechanism remains unclear. Vesicle budding from the Golgi in vitro is dependent on an 85-kDa cytosolic complex comprising p62 homologous to the regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase)35, and a small GTPase36. How these interactions mediate vesicle formation is not clear, but changes in lipid charge might promote the membrane curvature that is required for vesicle formation. Specifying correct delivery of transport vesicles to different membrane domains
Delivery of transport vesicles from the TGN to different plasma membrane domains has a remarkably high fidelity. Such fidelity could be based on vesicles targeting along the cytoskeleton, and/or by specifying vesicle docking and fusion with the correct membrane domain (Fig. 1). Surprisingly, the fidelity of vesicle transport from the TGN to either the apical or basallateral membrane domain requires neither an intact actin or microtubule cytoskeleton37. However, the efficiency of delivery is decreased when microtubules are depolymerized37, indicating that the cytoskeleton
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Cytoplasmic coat Cytoplasm
Basal–lateral membrane protein
Apical membrane protein
GPI-anchored protein Glycosphingolipid raft
Sorting signals Cytoplasmic domain Lumenal domain • Tyrosine-based (e.g. LDL-R) • Glycosylphosphatidylinositol (GPI) • Non-tyrosine based (e.g. pIgA-R) group • Di-hydrophobic (e.g. Fc-R) • Glycosylation (N-, O-glycan) Sorting mechanisms Clustering by cytoplasmic coat proteins (e.g. AP1/µ1B, p200/myosin II), RhoA GTPases
Partitioning into microdomains (e.g. lipid rafts, sorting lectins)
Sec6–Sec8 is recruited rapidly to sites of cell–cell contact and eventually organizes to the apical junctional complex. In MDCK cells permeabilized by streptolysin-O, antibodies against Sec8 inhibit delivery of LDL receptor to the basal-lateral membrane, but not p75NTR to the apical membrane43. It remains to be shown whether vesicle docking and fusion occur at the apical junctional region. These results indicate that lateral membrane recruitment of the Sec6–Sec8 complex is a consequence of cell–cell adhesion and is essential for the biogenesis of the surface polarity of epithelial cells. Presumably, the Sec6/8 complex interacts in some way with the SNARE machinery to deliver transport vesicles for fusion with the plasma membrane, but there is no experimental evidence for how this might occur.
TRENDS in Cell Biology
Protein sorting at the plasma membrane Fig. 2. Schematic representation of basallateral (left) and apical (right) transport vesicles budding from the trans Golgi network into the cytoplasm. Membrane proteins are sorted based on specific sorting signals (center) and sorting mechanisms (bottom) specific for either the basal-lateral (left) or apical pathways (right). Abbreviations: AP1, adaptor protein 1; Fc-R, crystalizable fragment receptor; LDL-R, lowdensity lipoprotein receptor.
Acknowledgements We apologize to our colleagues for omitting many references that detail the studies reported in this review owing to space constraints. Work from the Nelson laboratory is supported by a grant from the National Institutes of Health to W.J.N. (GM35527), and C.Y. was also supported by a Walter V. and Idun Y. Berry Fellowship.
does track vesicles to the plasma membrane but that the ultimate specification of docking and fusion is likely to reside in the interaction between the transport vesicle and target membrane domain (see below). Indeed, cytoskeleton motors are involved in post-TGN trafficking of vesicles to different membrane domains38. Mechanisms that specify vesicle docking and fusion with the apical and basal-lateral membrane have been examined within the conceptual framework of the SNARE hypothesis, which states the following: correct pairing of addressing proteins on the transport vesicle (termed v-SNAREs) with cognate receptors on the target membrane (termed t-SNAREs) determines the specificity of vesicle docking and fusion. Endogenous t-SNAREs, located on a target membrane, have polarized distributions in several epithelial cell types39. The t-SNARE syntaxin 3 is localized to the apical plasma membrane in a variety of cell types. By contrast, syntaxin 4 is expressed predominantly on the basal-lateral membrane domain. Less is known about the organization of v-SNAREs. However, treatment of permeabilized MDCK cells with either tetanus toxin or botulinum neurotoxin serotype F, which cleave the v-SNAREs VAMP-1, 2 and cellubrevin, significantly reduced the efficiency of vesicular stomatitis virus G protein transport to the basal-lateral membrane, but had no inhibitory effect on delivery of influenza virus hemagglutinin to the apical membrane40. This result suggests that v-SNAREs are not required for apical secretion. However, a toxininsensitive VAMP has been identified in Caco-2 cells, which forms a complex with the apical t-SNARE syntaxin-3 and SNAP-23 (Ref. 41, but see also Ref. 42). Recent studies indicate that a multiprotein complex (Sec6–Sec8) consisting of mammalian homologs of the yeast Sec3, Sec5, Sec6, Sec8, Sec10, Sec15 and Exo70 gene products is involved in specifying delivery of at least basal-lateral transport vesicles to the basal-lateral membrane domain. These Sec6/8 homologs are components of a cytosolic, ~17S complex in nonpolarized MDCK epithelial cells. Upon initiation of Ca2+-dependent cell–cell adhesion, about 70% of http://tcb.trends.com
Although a perception is that targeting of proteins from the TGN to specific membrane domains is sufficient to generate cell-surface polarity, it is clear that cells have developed additional mechanisms to control protein distributions at the plasma membrane. The plasma membrane is turned over rapidly by endocytosis, and proteins must either be excluded selectively from internalization or, following internalization, be resorted to the correct membrane domain. Assembly of the fodrin-based membrane skeleton might play a direct role in the early formation of a basal-lateral membrane domain at sites of cell–cell contact by directing the retention and accumulation of specific proteins that have affinity for the fodrin lattice. Several lines of evidence highlight the importance of interactions between cadherin–catenin complexes and the membrane skeleton in the localization of certain proteins, such as the Na+/K+ATPase, to sites of cell–cell adhesion. The concept of the membrane skeleton as a protein-sorting machine is attractive as it predicts that certain proteins, such as the Na+/K+-ATPase, would be preferentially excluded from endocytic pathways and would accumulate at cell–cell contact sites. By contrast, mistargeted membrane proteins would not be incorporated into the membrane skeleton and consequently would be internalized more rapidly. Data to support these predictions have been presented44. Proteins internalized in the endocytic pathway have several potential fates: delivery to the lysosome for degradation; recycling to the membrane domain of origin; or transcytosis to another membrane domain45 (Fig. 1). In hepatocytes, newly synthesized membrane proteins are delivered from the TGN to the basal-lateral (sinusoidal) membrane, from where apical proteins are selectively internalized by endocytosis and then delivered along microtubules to the apical (bile canaliculus) membrane. This process – termed transcytosis – also occurs in simple epithelia, as exemplified by the poly-IgA receptor45. However, it has been difficult to determine precisely the mechanisms involved in selection of
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proteins for transcytosis in hepatocytes, although the development of a robust cell-culture model – WIF-B cells – should aid this analysis considerably46. The next 10 years
One big difference in this field now, compared with 10 years ago, is that a greater variety of approaches, experimental systems and disciplines are focusing on epithelial cell polarity. Now, studies in Drosophila and Caenorhabditis elegans are asking how polarized epithelial cells develop in vivo and are using genetic screens to identify new components of signaling pathways that regulate the formation of epithelial sheets (for example, see Ref. 47). Physiologists are References 1 Almers, W. and Stirling, C. (1984) Distribution of transport proteins over animal cell membranes. J. Membr. Biol. 77, 169–186 2 Rodriguez-Boulan, E. and Nelson, W.J. (1989) Morphogenesis of the polarized epithelial cell phenotype. Science 245, 718–725 3 Simons, K. and Wandinger-Ness, A. (1990) Polarized sorting in epithelia. Cell 62, 207–210 4 Brewer, C.B. and Roth, M.G. (1991). A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin. J. Cell Biol. 114, 413–421 5 Mostov, K.E. et al. (1986) Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis. Cell 47, 359–364 6 Aroeti, B. et al. (1993) Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor. J. Cell Biol. 123, 1149–1160 7 Matter, K. et al. (1992) Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71, 741–753 8 Collawn, J.F. et al. (1990) Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell 63, 1061–1072 9 Casanova, J.E. et al. (1991) An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor. Cell 66, 65–75 10 Matter, K. et al. (1994) Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J. Cell Biol. 126, 991–1004 11 Le Gall, A.H. et al. (1997) The neural cell adhesion molecule expresses a tyrosine-independent basolateral sorting signal. J. Biol. Chem. 272, 4559–4567 12 Ohno, H. et al. (1995) Interaction of tyrosinebased sorting signals with clathrin-associated proteins. Science 269, 1872–1875 13 Rapoport, I.Y.C. et al. (1998) Dileucine-based sorting signals bind to the β chain of AP-1 at a site distinct and regulated differently from the tyrosinebased motif-binding site. EMBO J. 17, 2148–2155 14 Folsch, H. et al. (1999) A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99, 189–198 15 Folsch, H. et al. (2001) Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells. J. Cell Biol. 152, 595–606 16 Höning, S. et al. (1998) A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding http://tcb.trends.com
examining how protein trafficking and protein complexes at the plasma membrane regulate ion-channel and transporter expression, activity and distribution – the number of diseases that have as their basis a defect in membrane protein sorting is increasing (for example, polycystic kidney disease, Beckwith–Weidemann syndrome, Zellweger syndrome and Davidson’s syndrome). Finally, in vitro reconstitution assays are being used to ask important questions about the molecular machinery involved in protein sorting and packaging in the TGN and about how vesicles dock and fuse with specific membrane domains. The next ten years will be exciting.
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