Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.     

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.     

The clathrin adaptor AP-1A mediates basolateral polarity

Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.     

Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells.

The effects of brefeldin A (BFA) on transferrin (Tf) transcellular transport, Tf receptor (TfR) distribution, and TfR-mediated endocytosis in filter-grown Madin-Darby canine kidney (MDCK) cells were studied. BFA (1.6 micrograms/ml) markedly enhanced the transcytosis of 125I-labeled Tf (125I-Tf) in both apical-to-basal and basal-to-apical directions; yet, BFA did not enhance the transcytosis of either native horseradish peroxidase (HRP) or membrane-bound HRP-poly(L-lysine) conjugates. Furthermore, this enhanced transcytosis of 125I-Tf was abolished either by competition with excess unlabeled Tf or by incubation at temperatures less than or equal to 25 degrees C. In addition, BFA treatment to MDCK cells: (a) increased 125I-Tf specific binding to the apical membrane and decreased 125I-Tf specific binding to the basal membrane; (b) decreased TfR recycling at the basolateral membrane; (c) altered the apical/basolateral distribution of TfRs in favor of the apical side; and (d) markedly increased 59Fe extraction, but not transcytosis, from apically endocytosed 59Fe-loaded Tf. These effects are consistent with a model in which BFA alters the traffic pattern of internalized Tf by decreasing basolateral TfR recycling, while diverting the nonrecycled fraction to the apical side of the cell. Our results indicate that, unlike the reported inhibition of polymeric IgA transcytosis (Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617-627), BFA can enhance the transcytosis of Tf in MDCK cells. Thus, by altering the intracellular traffic of ligand-receptor complexes, BFA can elicit either a decrease or an increase in transcytosis depending on the nature of the intracellular receptor processing.     

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.     

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.     

Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism

We have evaluated transcytotic routes in MDCK cells for their ability to generate a polarized surface distribution of trafficking proteins by following the intracellular sorting of transferrin receptors (TRs). We find that the selective basolateral expression of TRs is maintained in the face of extensive trafficking between the apical and basolateral surfaces. Biochemical studies of receptors loaded with tracer under conditions approaching steady state indicate that TRs internalized from the two surfaces are extensively colocalized within MDCK cells and that both populations of receptors are selectively delivered to the basolateral surface. Tailless TRs in which the cytoplasmic domain has been deleted display an unpolarized cell surface distribution and recycle in an unpolarized fashion. We show by EM that wild-type receptors internalized from each surface are colocalized within endosomal elements distributed throughout the cytoplasm. By preloading endosomal elements directly accessible from the basolateral surface with transferrin (Tf)-HRP, we show that apically internalized TRs rapidly enter the same compartment. We also show that both transcytosing (apically internalized) and recycling (basolaterally internalized) TRs are delivered to the basolateral border by a distinctive subset of exocytotic, 60-nm-diam vesicles. Together, the biochemical and morphological data show that apical and basolateral endosomes of MDCK cells are interconnected and contain a signal- dependent polarized sorting mechanism. We propose a dynamic model of polarized sorting in MDCK cells in which a single endosome-based, signal-dependent sorting step is sufficient to maintain the polarized phenotype.     

Actin Dependence of Polarized Receptor Recycling in Madin-Darby Canine Kidney Cell Endosomes

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.     

Bidirectional transcytosis determines the steady state distribution of the transferrin receptor at opposite plasma membrane domains of BeWo cells

Trophoblast-like BeWo cells form well-polarized epithelial monolayers, when cultured on permeable supports. Contrary to other polarized cell systems, in which the transferrin receptor is found predominantly on the basolateral cell surface, BeWo cells express the transferrin receptor at both apical and basolateral cell surfaces (Cerneus, D.P., and A. van der Ende. 1991. J. Cell Biol. 114: 1149-1158). In the present study we have addressed the question whether BeWo cells use a different sorting mechanism to target transferrin receptors to the cell surface, by examining the biosynthetic and transcytotic pathways of the transferrin receptor in BeWo cells. Using trypsin and antibodies to detect transferrin receptors at the cell surface of filter-grown BeWo cells, we show that at least 80% of newly synthesized transferrin receptor follows a direct pathway to the basolateral surface, demonstrating that the transferrin receptor is efficiently intracellularly sorted. After surface arrival, pulse-labeled transferrin receptor equilibrates between apical and basolateral cell surfaces, due to ongoing transcytotic transport in both directions. The subsequent redistribution takes over 120 min and results in a steady state distribution with 1.5-2.0 times more transferrin receptors at the basolateral surface than at the apical surface. By monitoring the fate of surface-bound 125I-transferrin, internalized either from the apical or basolateral surface transcytosis of the transferrin receptor was studied. About 15% of 125I-transferrin is transcytosed in the basolateral to apical direction, whereas 25% is transcytosed in the opposite direction, indicated that the fraction of receptors involved in transcytosis is roughly twofold higher for the apical receptor pool, as compared to the basolateral pool. Upon internalization, both apical and basolateral receptor pools become redistributed on both surfaces, resulting in a twofold higher number of transferrin receptors at the basolateral surface. Our results indicate that in BeWo cells bidirectional transcytosis is the main factor in surface distribution of transferrin receptors on apical and basolateral surfaces, which may represent a cell type-specific, post-endocytic, sorting mechanism.     

The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non- transcytosing molecules.     

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.     

Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells.

The influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. By using deletion mutants and chimeric constructs of influenza virus NA with the human transferrin receptor, a type II basolateral transmembrane protein, we investigated the location of the apical sorting signal of influenza virus NA. When these mutant and chimeric proteins were expressed in stably transfected polarized MDCK cells, the transmembrane domain of NA, and not the cytoplasmic tail, provided a determinant for apical targeting in polarized MDCK cells and this transmembrane signal was sufficient for sorting and transport of the ectodomain of a reporter protein (transferrin receptor) directly to the apical plasma membrane of polarized MDCK cells. In addition, by using differential detergent extraction, we demonstrated that influenza virus NA and the chimeras which were transported to the apical plasma membrane also became insoluble in Triton X-100 but soluble in octylglucoside after extraction from MDCK cells during exocytic transport. These data indicate that the transmembrane domain of NA provides the determinant(s) both for apical transport and for association with Triton X-100-insoluble lipids.     

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.     

Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.     

Assessment of heterologous membrane protein polarity in transiently transfected MDCK cells

We have evaluated transient transfection of MDCK cells by the DEAE-dextran/chloroquine method as a rapid method for study of heterologous plasma membrane protein polarity. Transiently transfected cells reseeded onto permeable supports formed confluent monolayers with normal tight junctions and normal distribution of endogenous apical and basolateral surface markers. Transfected monolayers reseeded onto opaque polycarbonate filters attained cell heights 3 times greater than on transparent filters. Conventional and confocal immunofluorescence microscopy were used to assess polarity of transient expression of heterologous proteins previously defined in stably transfected cell lines as apical (DAF-CD55), basolateral (VSV-G), and nonpolarized (CD7) in distribution. Through each transiently expressed protein exhibited a polarity phenotype in most cells which resembled the stable phenotype, consistency of polarized localization was less than in stably transfected cells. Similar results were obtained by lipofection. We conclude that transient transfection of MDCK cells may be useful as a rapid screen, but is not sufficiently reliable for definitive assessment of heterologous membrane proein polarity.Abbreviations CD55-DAF CD55-Decay-accelearating factor - DMSO Dimethylsulfoxide - FBS Fetal bovine serum - FITC Fluorescein isothiocyanate - MDCK Madin Darby canine kidney cells - PBS Phosphate-buffered saline - TER Transepithelial resistance - VSVG Vesicular stomatis virus G protein     

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.     

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.     

Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medical recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.