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.     

Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant

In MDCK cells, Golgi to basolateral transport of several membrane proteins has been found to involve a cytoplasmic domain determinant. In some cases (Fc receptor, lysosomal glycoprotein Igp120), the determinant appears similar to that required for endocytosis via clathrin-coated pits; for Igp120, elimination of a single cytoplasmic domain tyrosine both blocks internalization and results in apical transport. In other cases (LDL receptor), the determinant does not involve the cytoplasmic domain tyrosine required for endocytosis. Thus, contrary to current models, basolateral transport in MCDK cells occurs not by default but depends on one or more cytoplasmic domain determinants, the precise nature of which is unknown. For some proteins, it is closely related to coated pit determinants. The fact that many membrane proteins can reach the apical surface in the absence of this determinant suggests that signals for apical transport are widely distributed.     

Hierarchy of sorting signals in chimeras of intestinal lactase-phlorizin hydrolase and the influenza virus hemagglutinin

Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur.     

Basolateral sorting signals differ in their ability to redirect apical proteins to the basolateral cell surface

Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting.     

A novel cellular phenotype for familial hypercholesterolemia due to a defect in polarized targeting of LDL receptor

Basolateral targeting of membrane proteins in polarized epithelial cells typically requires cytoplasmic domain sorting signals. In the familial hypercholesterolemia (FH)-Turku LDL receptor allele, a mutation of glycine 823 residue affects the signal required for basolateral targeting in MDCK cells. We show that the mutant receptor is mistargeted to the apical surface in both MDCK and hepatic epithelial cells, resulting in reduced endocytosis of LDL from the basolateral/sinusoidal surface. Consequently, virally encoded mutant receptor fails to mediate cholesterol clearance in LDL receptor-deficient mice, suggesting that a defect in polarized LDL receptor expression in hepatocytes underlies the hypercholesterolemia in patients harboring this allele. This evidence directly links the pathogenesis of a human disease to defects in basolateral targeting signals, providing a genetic confirmation of these signals in maintaining epithelial cell polarity.     

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.     

Identification of a basolateral membrane targeting signal within the cytoplasmic domain of myelin/oligodendrocyte glycoprotein

Oligodendrocytes possess two distinct membrane compartments--uncompacted plasma membrane (cell body, processes) and compact myelin. Specific targeting mechanisms must exist to establish and maintain these membrane domains. Polarized epithelial cells have the best characterized system for targeting components to apical and basolateral compartments. Since oligodendrocytes arise from neuroepithelial cells, we investigated whether they might utilize targeting paradigms similar to polarized epithelial cells. Myelin/oligodendrocyte glycoprotein (MOG) is a transmembrane Ig-like molecule restricted to uncompacted oligodendroglial plasma membrane. We stably expressed MOG in Madin-Darby canine kidney (MDCK) Type II epithelial cells, which have been extensively used in protein-targeting studies. Data from surface biotinylation assays and confocal microscopy revealed that MOG sorts exclusively to the basolateral membrane of MDCK cells. Expression vectors containing progressive truncations of MOG from the cytoplasmic C-terminus were expressed in MDCK cells to localize basolateral sorting signals. A loss of only four C-terminal residues results in some MOG expression at the apical surface. More strikingly, removal of the C-terminal membrane associated hydrophobic domain from MOG results in complete loss of basolateral sorting and specific targeting to the apical membrane. These data suggest that myelinating oligodendrocytes may utilize a sorting mechanism similar to that of polarized epithelia.     

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.     

Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants.

In MDCK cells, transport of membrane proteins to the basolateral plasma membrane has been shown to require a distinct cytoplasmic domain determinant. Although the determinant is often related to signals used for localization in clathrin-coated pits, inactivation of the coated pit domain in the human LDL receptor did not affect basolateral targeting. By expressing mutant and chimeric LDL receptors, we have now identified two independently acting signals that are individually sufficient for basolateral targeting. The two determinants mediate basolateral sorting with different efficiencies, but both contain tyrosine residues critical for activity. The first determinant was colinear with, but distinct from, the coated pit domain of the receptor. The second was found in the C-terminal region of the cytoplasmic domain of the receptor and, although tyrosine-dependent, did not mediate endocytosis. The results suggest that membrane proteins can have functionally redundant signals for basolateral transport and that a tyrosine-containing motif may be a common feature of multiple intracellular sorting events.     

Microtubule perturbation retards both the direct and the indirect apical pathway but does not affect sorting of plasma membrane proteins in intestinal epithelial cells (Caco-2).

Endogenous plasma membrane proteins are sorted from two sites in the human intestinal epithelial cell line Caco-2. Apical proteins are transported from the Golgi apparatus to the apical domain along a direct pathway and an indirect pathway via the basolateral membrane. In contrast, basolateral proteins never appear in the apical plasma membrane. Here we report on the effect of the microtubule-active drug nocodazole on the post-synthetic transport and sorting of plasma membrane proteins. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the appearance of three apical and one basolateral protein in plasma membrane domains. Nocodazole was found to drastically retard both the direct transport of apical proteins from the Golgi apparatus and the indirect transport (transcytosis) from the basolateral membrane to the apical cell surface. In contrast, neither the transport rates of the basolateral membrane nor the sorting itself were significantly affected by the nocodazole treatment. We conclude that an intact microtubular network facilitates, but is not necessarily required for, the transport of apical membrane proteins along the two post-Golgi pathways to the brush border.     

A hepatocyte-specific basolateral membrane protein is targeted to the same domain when expressed in Madin-Darby canine kidney cells

Different mechanisms for polarized sorting of apical and basolateral plasma membrane proteins appear to be operative in different cell types. In hepatocytes, all proteins are first transported to the basolateral surface, where sorting (probably signal-mediated) of apical proteins then takes place. In contrast, in Madin-Darby canine kidney (MDCK) cells, proteins are directly transported from the trans-Golgi network to their appropriate plasma membrane domain. In order to study the differences in the sorting requirements of the two cell types, we have expressed a hepatocyte-specific basolateral membrane protein, the asialoglycoprotein receptor H1, in MDCK cells. H1 was found to be specifically transported to the basolateral domain also in this heterologous system, suggesting that either the same basolateral targeting signal is operative in both cell types or, more likely, that basolateral transport occurs "by default," i.e. without the requirement for a sorting signal.     

Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.     

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored protein, is localized on both the apical and basolateral domains of polarized Madin-Darby canine kidney cells.

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor and N-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane gamma-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4-8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells.     

N-glycans are not a universal signal for apical sorting of secretory proteins.

In MDCK cells, N-glycans have been shown to determine the sorting of secretory proteins and membrane proteins to the apical domain in the absence of a dominant basolateral targeting signal. We have examined the sorting of endogenous proteins in ECV304 cells in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. A prominent apically secreted protein of 71 kDa was not N-glycosylated and continued to be secreted apically in the presence of tunicamycin. In contrast, other endogenous proteins that were N-glycosylated were secreted preferentially into the basolateral medium or without polarity. When rat growth hormone was expressed in MDCK and ECV304 cells, we observed 65 and 94% of the secretion to the basolateral medium, respectively. Introduction of a single N-glycan caused 83% of the growth hormone to be secreted at the apical surface in MDCK cells but had no significant effect on the polarity of secretion of growth hormone in ECV304 cells. These results indicate that not all cell lines recognise N-glycans as a signal for apical sorting and raises the possibility of using ECV304 cells as a model system for analysis of apical sorting molecules.     

Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information.

Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.     

Trafficking of immunoglobulin receptors in epithelial cells:signals and cellular factors

A typical feature of epithelial cells is the polarized distribution of their respective plasma membrane proteins. Apical and basolateral proteins can be sorted both in the trans-Golgi network and endosomes, or in both locations. Inclusion into basolateral carriers in the TGN requires the presence of distinct cytoplasmic determinants, which also appear to be recognized in endosomes. Inactivation of the basolateral sorting information leads to the efficient apical delivery, probably due to the unmasking of a recessive apical signal. Factors associated with the cytosolic face of organelles probably not only recognize these signals to mediate the inclusion of the proteins into the correct transport vesicles, but also target the carriers to the corresponding plasma membrane domain. Our interest has focused on analyzing at the molecular level how epithelial MDCK cells generate and maintain a polarized phenotype, taking advantage of immunoglobulin receptors to study the biosynthetic and endocytic pathways and the corresponding sorting events.     

The basolateral targeting signal of CD147 (EMMPRIN) consists of a single leucine and is not recognized by retinal pigment epithelium

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.     

A Secretory Golgi Bypass Route to the Apical Surface Domain of Epithelial MDCK Cells

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae . We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.     

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.     

Cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells

Although it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin-Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans-Golgi network with a halftime of approximately 45 min and have an approximate t_1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg-651 were localized predominantly at the apical surface of filter-grown cells, whereas receptors truncated at Leu-723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise loacation of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states. © 1995 Wiley-Liss, Inc.