Polarized apical sorting of guanylyl cyclase C is specified by a cytosolic signal

Receptor guanylyl cyclases respond to ligand stimulation by increasing intracellular cGMP, thereby initiating a variety of cell-signaling pathways. Furthermore, these proteins are differentially localized at the apical and basolateral membranes of epithelial cells. We have identified a region of 11 amino acids in the cytosolic COOH terminus of guanylyl cyclase C (GCC) required for normal apical localization in Madin-Darby canine kidney (MDCK) cells. These amino acids share no significant sequence homology with previously identified cytosolic apical sorting determinants. However, these amino acids are highly conserved and are sufficient to confer apical polarity to the interleukin-2 receptor alpha-chain (Tac). Additionally, we find two molecular weight species of GCC in lysates prepared from MDCK cells over-expressing GCC but observe only the fully mature species on the cell surface. Using pulse-chase analysis in polarized MDCK cells, we followed the generation of this mature species over time finding it to be detectable only at the apical cell surface. These data support the hypothesis that selective apical sorting can be determined using short, cytosolic amino acid motifs and argue for the existence of apical sorting machinery comparable with the machinery identified for basolateral protein traffic.     

Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization

The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75–green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery.     

The rat liver Na(+)/bile acid cotransporter. Importance of the cytoplasmic tail to function and plasma membrane targeting

To understand the potential functions of the cytoplasmic tail of Na(+)/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta-turn unfavorable tetrapeptide Y(321)KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta-turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins.     

Sorting of H,K-ATPase beta-subunit in MDCK and LLC-PK cells is independent of mu 1B adaptin expression

The cytoplasmic tail of the H,K-ATPase beta-subunit contains a putative tyrosine-based motif that directs the beta-subunit's basolateral sorting when it is expressed in Madin-Darby Canine Kidney (MDCK) cells. When expressed in LLC-PK(1) cells, however, the beta-subunit is localized to the apical membrane. Several proteins that contain tyrosine-based motifs, including the low-density lipoprotein and transferrin receptors, show a similar sorting 'defect' when expressed in LLC-PK(1) cells. For low-density lipoprotein and transferrin receptors, this behavior is due to the differential expression of the mu 1B subunit of the AP-1B clathrin adaptor complex. mu 1B is expressed by MDCK cells, but not LLC-PK(1) cells, and transfection of mu 1B into LLC-PK(1) cells restores basolateral localization of low-density lipoprotein and transferrin receptors. For the beta-subunit, however, mu B expression in LLC-PK(1) cells does not induce its basolateral expression. We found that the beta-subunit interacts with both mu 1B and mu 1A in vitro and in vivo. The capacity to participate in a mu 1B interaction therefore is not sufficient to program the beta-subunit's basolateral localization in MDCK cells. Our data suggest that the H,K-ATPase beta-subunit's basolateral sorting signal is either masked in certain epithelial cells, or requires an interaction with sorting machinery other than AP-1B for delivery to the basolateral plasma membrane.     

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

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

Targeting of transmembrane protein shrew-1 to adherens junctions is controlled by cytoplasmic sorting motifs

We recently identified transmembrane protein shrew-1 and showed that it is able to target to adherens junctions in polarized epithelial cells. This suggested shrew-1 possesses specific basolateral sorting motifs, which we analyzed by mutational analysis. Systematic mutation of amino acids in putative sorting signals in the cytoplasmic domain of shrew-1 revealed three tyrosines and a dileucine motif necessary for basolateral sorting. Substitution of these amino acids leads to apical localization of shrew-1. By applying tannic acid to either the apical or basolateral part of polarized epithelial cells, thereby blocking vesicle fusion with the plasma membrane, we obtained evidence that the apically localized mutants were primarily targeted to the basolateral membrane and were then redistributed to the apical domain. Further support for a postendocytic sorting mechanism of shrew-1 was obtained by demonstrating that mu1B, a subunit of the epithelial cell-specific adaptor complex AP-1B, interacts with shrew-1. In conclusion, our data provide evidence for a scenario where shrew-1 is primarily delivered to the basolateral membrane by a so far unknown mechanism. Once there, adaptor protein complex AP-1B is involved in retaining shrew-1 at the basolateral membrane by postendocytic sorting mechanisms.     

An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor

The polymeric immunoglobulin receptor is normally delivered from the Golgi to the basolateral surface of epithelial cells and then transports polymeric IgA and IgM to the apical surface. We now report that a 14 residue segment of the 103 amino acid cytoplasmic domain, proximal to the plasma membrane, directs the receptor to the basolateral surface. A mutant receptor lacking these 14 amino acids is sorted directly to the apical surface from the Golgi. Furthermore, this sequence is sufficient to redirect an apical membrane protein, placental alkaline phosphatase, to the basolateral plasma membrane. We conclude that this sequence contains an autonomous signal, which specifies sorting from the Golgi to the basolateral surface, a process previously postulated to occur by default.     

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

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

unc-44 Ankyrin and stn-2 gamma-syntrophin regulate sax-7 L1CAM function in maintaining neuronal positioning in Caenorhabditis elegans

The L1 family of single-pass transmembrane cell adhesion molecules (L1CAMs) is conserved from Caenorhabditis elegans and Drosophila to vertebrates and is required for axon guidance, neurite outgrowth, and maintenance of neuronal positions. The extracellular region of L1CAMs mediates cell adhesion via interactions with diverse cell-surface and extracellular matrix proteins. In contrast, less is known regarding the function of the intracellular domains in the L1CAM cytoplasmic tail. Previously, we identified a role of the C. elegans L1CAM homolog, SAX-7, in maintaining neuronal and axonal positioning. Here, we demonstrate that this function is dependent on three conserved motifs that reside in the SAX-7 cytoplasmic tail: (1) the FERM-binding motif, (2) the ankyrin-binding domain, and (3) the PDZ-binding motif. Furthermore, we provide molecular and genetic evidence that UNC-44 ankyrin and STN-2 γ-syntrophin bind SAX-7 via the respective ankyrin-binding and PDZ-binding motifs to regulate SAX-7 function in maintaining neuronal positioning.     

An extracellular loop of the human non-gastric H,K-ATPase alpha-subunit is involved in apical plasma membrane polarization.

The human non-gastric H,K-ATPase, ATP1AL1, belongs to the gene family of P-type ATPases. Consistent with their physiological roles in ion transport, members of this group, including the Na,KATPase and the gastric and non-gastric H,K-ATPases, are differentially polarized to either the basolateral or apical plasma membrane in epithelial cells. However, their polarized distribution is highly complex and depends on specific sorting signals or motifs which are recognized by the subcellular targeting machinery. For the gastric H,K-ATPase it has been suggested that the 4(th) transmembrane spanning domain (TM4) and its flanking regions induce conformational sorting motifs which direct the ion pump exclusively to the epithelial apical membrane. Here, we show in transfected Madin-Darby canine kidney (MDCK) cells that the related non-gastric H,KATPase, ATP1AL1, does contain similar sorting motifs in close proximity to TM4. A short extracellular loop between TM3 and TM4 is critical for this pump's apical delivery. A single point mutation in the corresponding region redirects ATP1AL1 to the basolateral membrane. In conclusion, our work provides further evidence that the cellular distribution of P-type ATPases is determined by conformational sorting motifs.     

Identification of a Cytoplasmic Signal for Apical Transcytosis

Polarized epithelial cells contain apical and basolateral surfaces with distinct protein compositions. To establish and maintain this asymmetry, newly made plasma membrane proteins are sorted in the trans Golgi network for delivery to apical or basolateral surfaces. Signals for basolateral sorting are generally located in the cytoplasmic domain of the protein, whereas signals for apical sorting can be in any part of the protein and can depend on N-linked glycosylation of the protein. Signals for constitutive transcytosis to the apical surface have not been reported. In this study, we used the polymeric immunoglobulin receptor (pIgR), which is biosynthetically delivered to the basolateral surface. There the pIgR can bind a ligand and, with or without bound ligand, the pIgR can then be transcytosed to the apical surface. We found that the glycosylation of the pIgR did not affect the biosynthetic transport of the pIgR. However, glycosylation had an effect on pIgR apical transcytosis. Importantly, analysis of the cytoplasmic tail of the pIgR suggested that a short peptide segment was sufficient to transcytose the pIgR or a neutral reporter from the basolateral to the apical surface. This apical transcytosis sorting signal was not involved in polarized biosynthetic traffic of the pIgR.     

The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane

To perform vectorial secretory and transport functions that are critical for the survival of the organism, epithelial cells sort plasma membrane proteins into polarized apical and basolateral domains. Sorting occurs post-synthetically, in the trans Golgi network (TGN) or after internalization from the cell surface in recycling endosomes, and is mediated by apical and basolateral sorting signals embedded in the protein structure. Basolateral sorting signals include tyrosine motifs in the cytoplasmic domain that are structurally similar to signals involved in receptor internalization by clathrin-coated pits. Recently, an epithelial-specific adaptor protein complex, AP1B, was identified. AP-1B recognizes a subset of basolateral tyrosine motifs through its mu 1B subunit. Here, we characterized the post-synthetic and post-endocytic sorting of the fast recycling low density lipoprotein receptor (LDLR) and transferrin receptor (TfR) in LLC-PK1 cells, which lack mu 1B and mis-sort both receptors to the apical surface. Targeting and recycling assays in LLC-PK1 cells, before and after transfection with mu 1B, and in MDCK cells, which express mu 1B constitutively, suggest that AP1B sorts basolateral proteins post-endocytically.     

Organization of vesicular trafficking in epithelia

Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for apical and basolateral plasma-membrane proteins, and have identified components that guide apical and basolateral proteins along these pathways. These components include apical and basolateral sorting signals, adaptors for basolateral signals, and docking and fusion proteins for vesicular trafficking. Recent live-cell-imaging studies provide a real-time view of sorting processes in epithelial cells, including key roles for actin, microtubules and motors in the organization of post-Golgi trafficking.     

Identification of an apical sorting determinant in the cytoplasmic tail of megalin

Megalin is the main endocytic receptor ofthe proximal tubule and is responsible for reabsorption of manyfiltered proteins. In contrast to other members of the low-densitylipoprotein (LDL) receptor gene family, it is expressed on the apicalplasma membrane (PM) of polarized epithelial cells. To identifymegalin's apical sorting signal, we generated deletion mutants andchimeric minireceptors composed of complementary regions of megalin andLDL receptor-related protein (LRP) and assessed the distribution of themutants in Madin-Darby canine kidney (MDCK) cells by immunofluorescenceand cell surface biotinylation. Megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, ofMDCK cells. We found that the information that directs apical sortingis present in the cytoplasmic tail (CT) of megalin, which containsthree NPXY motifs, YXXØ, SH3, and dileucine motifs, and a PDZ-bindingmotif at its COOH terminus. Deletion analysis established that aminoacids 107-136 of the megalin-CT containing the second NPXY-likemotif are critical for apical sorting and targeting, whereas theregions containing the first and third NPXY motifs are required forefficient endocytosis. We conclude that the megalin-CT contains a novelapical sorting determinant and that cytoplasmic sorting machineryexists in MDCK cells for some apical transmembrane proteins.

     

The apical sorting signal for human GLUT9b resides in the N-terminus

The two splice variants of human glucose transporter 9 (hGLUT9) are targeted to different polarized membranes. hGLUT9a traffics to the basolateral membrane, whereas hGLUT9b traffics to the apical region. This study examines the sorting mechanism of these variants, which differ only in their N-terminal domain. Mutating a di-leucine motif unique to GLUT9a did not affect targeting. Chimeric proteins were made using GLUT1, a basolaterally targeted transporter, and GLUT3, an apically targeted protein whose signal lies in the C-terminus. Overexpression of the chimeric proteins in polarized cells demonstrates that the N-terminus of hGLUT9b contains a signal capable of redirecting GLUT1 to the apical membrane. The N-terminus of hGLUT9a, however, does not contain a basolateral signal sufficient enough to redirect GLUT3. Portions of the GLUT9a N-terminus were substituted with corresponding portions of the GLUT9b N-terminus to determine the motif responsible for apical targeting. The first 16 amino acids were not found to be a sufficient apical signal. The last ten amino acids of the N-termini differ only in amino-acid class at one location. In the B-form, leucine, a hydrophobic residue, is substituted for lysine, a basic residue, found in the A-form. However, mutation of the leucine in hGLUT9b to a lysine resulted in retention of the apical signal. We therefore believe the apical signal exists as an interplay between the final ten amino acids of the N-terminus and another motif within the protein such as the intracellular loop or other motifs within the N-terminus.     

A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase

The H,K-adenosine triphosphatase (ATPase) of gastric parietal cells is targeted to a regulated membrane compartment that fuses with the apical plasma membrane in response to secretagogue stimulation. Previous work has demonstrated that the alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta subunit carries the signal responsible for the cessation of acid secretion through the retrieval of the pump from the surface to the regulated intracellular compartment. By analyzing the sorting behaviors of a number of chimeric pumps composed of complementary portions of the H, K-ATPase alpha subunit and the highly homologous Na,K-ATPase alpha subunit, we have identified a portion of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral Na,K-ATPase to the apical surface in transfected epithelial cells. This motif resides within the fourth of the H,K-ATPase alpha subunit's ten predicted transmembrane domains. Although interactions with glycosphingolipid-rich membrane domains have been proposed to play an important role in the targeting of several apical membrane proteins, the apically located chimeras are not found in detergent-insoluble complexes, which are typically enriched in glycosphingolipids. Furthermore, a chimera incorporating the Na, K-ATPase alpha subunit fourth transmembrane domain is apically targeted when both of its flanking sequences derive from H,K-ATPase sequence. These results provide the identification of a defined apical localization signal in a polytopic membrane transport protein, and suggest that this signal functions through conformational interactions between the fourth transmembrane spanning segment and its surrounding sequence domains.     

PDZ-binding motifs are unable to ensure correct polarized protein distribution in the absence of additional localization signals

The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. To determine the specificity of the PDZ-binding motifs in establishing cellular distribution, we utilized a 111-amino acid region from the C-terminus of cystic fibrosis transmembrane conductance regulator (CFTR) that is able to direct apical localization of fused reporter proteins. Substitution of the C-terminal PDZ-binding motif of CFTR with corresponding motifs necessary for basolateral localization of other membrane proteins did not lead to the redistribution of the fusion protein to the basolateral membrane. Instead, some fusion proteins remained localized to the apical membrane, whereas others showed no specific distribution. The specificity of the PDZ-based interactions was substantially increased when specific amino acids located upstream of the classical PDZ-binding motifs were included. However, even the presence of a longer C-terminal motif from a basolateral protein could not ensure basolateral distribution of the fusion protein. Our results indicate that the C-terminal PDZ-binding motifs are not the primary signals for polarized protein distribution, although they are required for targeting and/or stabilization of protein at the given location.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2)

We studied the postsynthetic sorting of endogenous plasma membrane proteins in a polarized epithelial cell line, Caco-2. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the arrival of three apical and one basolateral protein at the apical and basolateral cell surface. Apical proteins were inserted simultaneously into both membrane domains. The fraction targeted to the basolateral domain was different for the three apical proteins and was subsequently sorted to the apical domain by transcytosis at different rates. In contrast, a basolateral protein was found in the basolateral membrane only. Thus, sorting of plasma membrane proteins occurred from two sites: the Golgi apparatus and the basolateral membrane. These data explain apparently conflicting results of earlier studies.     

A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells.

An important function of the low affinity IgG Fc receptor FcRII-B2 (FcR) on macrophages is the internalization of soluble antigen-antibody complexes for lysosomal degradation. Most endocytic receptors possess tyrosine-containing cytoplasmic determinants required for endocytosis. In many proteins, signals which overlap with the endocytosis determinant and share the same critical tyrosine residue also mediate basolateral sorting in the trans-Golgi network of epithelial cells. Despite the presence of two tyrosine residues in the FcR cytosolic domain, neither one is absolutely required for coated pit localization or basolateral targeting. Nevertheless, a short domain of 13 residues containing one of the non-critical tyrosine residues mediates endocytosis and basolateral delivery. Alanine scan mutagenesis of this region now revealed a critical role of a leucine-leucine motif in both events. These findings suggest that endocytosis and basolateral sorting can be mediated by both tyrosine- and di-leucine-based signals and confirm the close relationship between the two determinants already observed for 'classical' tyrosine-dependent motifs.