N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.

Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.     

Targeting of an apical endosomal protein to endosomes in Madin-Darby canine kidney cells requires two sorting motifs

The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin–Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site-directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin-positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate-binding protein rab11, and do not tubulate in response to low concentrations of brefeldin-A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule.     

Sialoforms of dipeptidylpeptidase IV from rat kidney and liver

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found in the cell surface membrane of many tissues, was employed as a model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.     

Apical cell surface expression of rat dipeptidyl peptidase IV in transfected Madin-Darby canine kidney cells.

Dipeptidyl peptidase IV (DPPIV) is a type II membrane glycoprotein that is predominantly localized to the apical plasma membrane in various epithelial cells. In order to understand in more detail the biogenesis and sorting of DPPIV, the cDNA for rat DPPIV was inserted into a mammalian plasmid expression vector so that DPPIV expression was driven by a control region composed of the SV40 early promoter region fused to the enhancer of the Rous sarcoma virus. Madin-Darby canine kidney cells transfected with this construct were found to express the DPPIV protein. In these transfected cells, the majority of DPPIV was present on the apial cell surface. This observation suggests that the information for apical surface localization is inherent in the DPPIV molecule itself and that this sorting information is decipherable in the epithelial cells of a different species. DPPIV is transported efficiently from the endoplasmic reticulum to the Golgi apparatus as assessed by pulse-chase experiments. Furthermore, evidence is presented which suggests that the majority of DPPIV is sorted intracellularly to the apical cell surface. The same protein has, however, been reported to be sorted by an indirect pathway through transcytosis from the basolateral to the apical cell surface in hepatocytes (Bartles, J.R., Feracci, H., M., Stinger, B., and Hubbard, A.L. (1987) J. Cell Biol. 105, 1241-1251). This study suggests that the same protein can take two different pathways in different cell types for its correct apical cell surface localization.     

Mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells

Enteropeptidase, a type II transmembrane protein of the enterocyte brush border, is sorted directly to the apical membrane of Madin-Darby canine kidney II cells. Apical targeting appears to be mediated by an N-terminal segment that contains a 27-amino acid residue O-glycosylated mucin-like domain consisting of two short mucin-like repeats, A and B. Targeting signals within these repeats were characterized by using green fluorescent protein (GFP) as a reporter. Constructs with a cleavable signal peptide and both repeats A and B were secreted apically. Similar constructs lacking mucin repeats were secreted randomly. Either repeat A or B was sufficient to direct apical targeting of GFP. O-linked oligosaccharides alone were not sufficient for targeting because fusion to a different O-glycosylated motif did not alter the random secretion of GFP, and several constructs with mutations in either repeat A or B were O-glycosylated and secreted randomly. In addition, repeat B appears to contain an apical targeting signal that functions in the absence of glycosylation. Density gradient centrifugation indicated that, unlike several other apically targeted membrane and soluble proteins, apical sorting of mucin-GFP chimeric proteins does not appear to utilize lipid rafts.     

Subcellular trafficking of exogenously expressed interferon-beta in Madin-Darby canine kidney cells

We have recently demonstrated that when IFN-beta was exogenously expressed in epithelial cells, transiently expressed IFN-beta was predominantly secreted from the cell side to which the transfection was performed, while stably expressed one was almost equally secreted to the apical and basolateral sides. In the present study, we analyzed the subcellular transport of IFN-beta using confocal imaging with green fluorescent protein (GFP)-tagged IFN-beta in Madin-Darby canine kidney (MDCK) cells. Stably expressed and transiently expressed human IFN-beta (HuIFN-beta)-GFPs were seen in upper regions of the nucleus. In stable HuIFN beta-GFP-producing transformants, transiently expressed mouse IFN-beta (MuIFN-beta) was apparently co-localized with the bulk of the constitutive HuIFN beta-GFP proteins at TGN, and a significant quantity of them then appeared to pass into distinct post-TGN vesicles, accepting either type of IFN. Meanwhile, when cells were co-transfected with both expression vectors, transiently expressed both IFNs tended to co-localize not only at TGN but in post-TGN vesicles. These results suggest that stably and transiently expressed IFN-betas, albeit co-localized at TGN, were transported through apparently discriminated post-TGN routes.     

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.     

Polarized expression of functional rat liver asialoglycoprotein receptor in transfected Madin-Darby canine kidney cells

The rat liver asialoglycoprotein receptor or rat hepatic lectin (RHL) consists of two polypeptide species, a major one designated RHL-1 and a minor one designated RHL-2/3, which exists in two differentially glycosylated forms. We have studied the biosynthesis, targeting, and function of the different forms after transfection of their cDNAs into the polarized Madin-Darby canine kidney cell line. In cells expressing only RHL-1, newly synthesized protein undergoes rapid intracellular degradation and is not detected at the cell surface. In contrast, RHL-2/3 when transfected alone is much more stable and is expressed at the basolateral surface of fiber-grown cells. When both forms are expressed together, newly synthesized RHL-1 escapes rapid degradation and is detected at the basolateral surface. In double transfectants a functional receptor is formed that specifically endocytoses and degrades ligand at the basolateral side.     

Heterogeneity of the tumorigenic phenotype expressed by Madin-Darby canine kidney cells

The mechanisms by which cells spontaneously immortalized in tissue culture develop the capacity to form tumors in vivo likely embody fundamental processes in neoplastic development. The evolution of Madin-Darby canine kidney (MDCK) cells from presumptively normal kidney cells to immortalized cells that become tumorigenic represents an example of neoplastic development in vitro. Studies of the mechanisms by which spontaneously immortalized cells develop the capacity to form tumors would benefit from quantitative in vivo assays. Most mechanistic correlations are evaluated by using single-dose tumor-induction experiments, which indicate only whether cells are or are not tumorigenic. Here we used quantitative tumorigenicity assays to measure dose-and time-dependent tumor development in nude mice of 3 lots of unmodified MDCK cells. The results revealed lot-to-lot variations in the tumorigenicity of MDCK cells, which were reflected by their tumor-inducing efficiency (threshold cell dose represented by mean tumor-producing dose; log(10) 50% endpoints of 5.2 for vial 1 and 4.4 for vial 2, and a tumor-producing dose of 5.8 for vial 3) and mean tumor latency (vial 1,6.6 wk; vial 2,2.9 wk; and vial 3,3.8 wk). These studies provide a reference for further characterization of the MDCK cell neoplastic phenotype and may be useful in delineating aspects of neoplastic development in vitro that determine tumor-forming capacity. Such data also are useful when considering MDCK cells as a reagent for vaccine manufacture.     

Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929-942), only small ( approximately 100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly ( approximately 10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits.Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.     

MAL mediates apical transport of secretory proteins in polarized epithelial Madin-Darby canine kidney cells.

The MAL proteolipid is an integral membrane protein identified as a component of the raft machinery for apical sorting of membrane proteins in Madin-Darby canine kidney (MDCK) cells. Previous studies have implicated lipid rafts in the transport of exogenous thyroglobulin (Tg), the predominant secretory protein of thyroid epithelial cells, to the apical surface in MDCK cells. We have examined the secretion of recombinant Tg and gp80/clusterin, a major endogenous secretory protein not detected in Triton X-100 insoluble rafts, for the investigation of the involvement of MAL in the constitutive apical secretory pathway of MDCK cells. We show that MAL depletion impairs apical secretion of Tg and causes its accumulation in the Golgi. Cholesterol sequestration, which blocks apical secretion of Tg, did not alter the levels of MAL in rafts but created a block proximal to Tg entrance into rafts. Apical secretion of gp80/clusterin was also inhibited by elimination of endogenous MAL. Our results suggest a role for MAL in the transport of both endogenously and exogenously expressed apical secretory proteins in MDCK cells.     

Ion channels in Madin-Darby canine kidney cells.

Ion channels in Madin-Darby canine kidney cells serve transepithelial chloride transport and probably cell volume regulation. Three distinct potassium channels and one anion channel have been revealed by patch clamp studies in Madin-Darby canine kidney cells. The potassium channels are activated by an increase in intracellular calcium activity. A number of hormones activate the potassium channels by an increase in intracellular calcium activity. However, under certain conditions the hormones hyperpolarize the cell membrane without increasing intracellular calcium activity sufficiently to activate the calcium-sensitive potassium channels. Thus, the hormones may activate potassium channels via another, as yet undefined, intracellular mechanism. The anion channel is stimulated by cAMP. Another factor modifying channel activity is cell volume: cell swelling leads probably to subsequent activation of potassium and anion channels. The net result is a variable transient hyperpolarization followed by a sustained depolarization of the cell membrane.     

rab4 regulates transport to the apical plasma membrane in Madin-Darby canine kidney cells.

The small GTPase rab4 is associated with early endosomes and regulates membrane recycling in fibroblasts. rab4 is present in epithelial cells; however, neither its localization nor function has been established in this cell type. We transfected Madin-Darby canine kidney cells with rab4, the GTPase-deficient mutant rab4Q67L, and the dominant negative mutant rab4S22N that poorly binds guanine nucleotides. Confocal immunofluorescence microscopy showed that rab4 was concentrated on internal structures at the lateral side of the cell around the nucleus. Quantitative immunoelectron microscopy revealed that the majority of rab4 was localized in the upper third of the cytoplasm. In cell surface binding experiments with (125)I-transferrin, we found a redistribution of transferrin receptor from the basolateral to the apical plasma membrane in cells expressing rab4 and rab4Q67L. After accumulation of transferrin at 16 degrees C in basolateral early endosomes, rab4 and rab4Q67L increased the amount of apically targeted transferrin receptor. A qualitatively similar effect was obtained in control cells treated with brefeldin A. The effects of brefeldin A and rab4 on apical targeting of transferrin receptor were not additive, suggesting that brefeldin A and rab4 may act in the same transport pathway from common endosomes.     

MAL regulates clathrin-mediated endocytosis at the apical surface of Madin-Darby canine kidney cells

MAL is an integral protein component of the machinery for apical transport in epithelial Madin-Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.     

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.     

Protein core-dependent glycosaminoglycan modification and glycosaminoglycan-dependent polarized sorting in epithelial Madin-Darby canine kidney cells

The proteoglycan serglycin (SG) fused to green fluorescent protein (GFP) is secreted predominantly from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cell monolayers, but the minor fraction secreted basolaterally carries more intensely sulfated glycosaminoglycan (GAG) chains (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem 280: 29596-29603). To investigate whether the domain with GAG attachment sites in SG (i) is sufficient to drive apical protein sorting and (ii) independently generates the sulfation differences observed in the apical and basolateral pathways, the GAG domain of SG was fused into the junction of rat growth hormone (rGH) and GFP and expressed in MDCK cells, either with or without two N-glycosylation sites in the rGH part. Both variants acquired chondroitin sulfate GAG chains and were secreted predominantly to the apical medium, to the same extent as rGH-GFP with two N-glycosylation sites only, and different from the nonsorted variant lacking glycosylation sites. Transfer of the GAG attachment domain from SG to the new rGH context abolished the differences in sulfation intensity and positions observed for SG in the apical and basolateral secretory routes. Thus, these differences are coded by elements outside the GAG attachment domain.     

The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.     

Endocytosis in filter-grown Madin-Darby canine kidney cells

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three- dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.     

Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.     

Differences in the apical and basolateral pathways for glycosaminoglycan biosynthesis in Madin-Darby canine kidney cells

Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.