Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical membrane of Madin-Darby canine kidney cells. II. Involvement of the Golgi complex

In the preceding paper (Pesonen M., W. Ansorge, and K. Simons, 1984, J. Cell Biol., 99:796-802), we have shown that transcellular transport of the membrane glycoprotein G of vesicular stomatitis virus implanted into the apical membrane of Madin-Darby canine kidney cells is transcytosed through the endosomal compartment to the basolateral plasma membrane. To determine whether the Golgi complex was involved in this process, G protein lacking sialic acid or all of the terminal sugars was implanted into the apical membrane and allowed to move to the basolateral membrane. Using the criteria of endoglycosidase H sensitivity, binding to Ricinus communis agglutinin and two-dimensional gel electrophoresis, the sugars on the transcytosed G protein were found to be the same as in the starting material. The absence of any involvement of the Golgi complex in transcytosis was supported by subcellular fractionation studies in which transcytosing G protein was never found in fractions containing galactosyl transferase.     

Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome

We have used temperature and nocodazole blocks in an in vivo basolateral to apical transcytosis assay to dissociate the early transcytotic steps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([Fab]2 fragment of IgG against the receptor's ectodomain) at 17 degrees C but do not deliver it to the apical PM. Instead, the ligand accumulates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule- requiring translocation step. We have characterized this compartment and show that it is distinct from basolateral transferrin recycling endosomes, basolateral early endosomes or late endosomes or lysosomes. The apical transcytotic compartment colocalizes with the compartment containing apically recycling membrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid phase marker (BSA). This compartment is an intermediate station of the overall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17 degrees C are subsequently incubated at 37 degrees C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of trimeric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gs alpha, to cells preloaded with transcytotic ligand at 17 degrees C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and function of the apical endosome are not affected since neither BFA nor cholera toxin inhibit the recycling of apically internalized membrane markers (ricin and pIgA-R ligand) from the same compartment. The data suggest that transcytosis connects the "membrane-sorting" sub-domain of the basolateral endosome with a homologous sub-domain of the apical endosome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, distinct from that used for recycling of apically internalized proteins (ricin or pIgA-R).     

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.     

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

Transcytotic efflux from early endosomes is dependent on cholesterol and glycosphingolipids in polarized hepatic cells

We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the "indirect" route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.     

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.     

Mathematical analysis of hepatic high density lipoprotein transport based on quantitative imaging data

Hepatocytes internalize high density lipoprotein (HDL) at the basolateral membrane. Most HDL is recycled while some is shuttled to the canalicular membrane by transcytosis. Here, transport of HDL was analyzed by mathematical modeling based on measurements in polarized hepatic HepG2 cells. Recycling of HDL from basolateral sorting endosomes was modeled by applying the rapid equilibrium approach. Analytical expressions were derived, which describe in one model the transport of HDL to the subapical compartment/apical recycling compartment, the biliary canaliculus (BC), and to late endosomes and lysosomes (LE/LYS). Apical endocytosis of HDL predicted by the model was confirmed for rhodamine-dextran and fluorescent asialoorosomucoid, markers for LE/LYS in living HepG2 cells. Budding of endocytic vesicles from the BC was directly observed by time lapse imaging of a fluorescent lipid probe. Based on fitted kinetic parameters and their covariance matrix a Monte Carlo simulation of HDL transport in hepatocytes was performed. The model was used to quantitatively assess release of HDL-associated free cholesterol by scavenger receptor BI. It is shown that only 6% of HDL-associated sterol reaches the BC as a constituent of the HDL particles, whereas the remaining sterol is rapidly released from HDL and shuttled to the BC by non-vesicular transport.     

Iron Imports. V. Transport of iron through the intestinal epithelium

Iron absorption across the brush-border membrane requires divalent metal transporter 1 (DMT1), whereas ferroportin (FPN) and hephaestin are required for exit across the basolateral membrane. However, how iron passes across the enterocyte is poorly understood. Both chaperones and transcytosis have been postulated to account for intracellular iron transport. With iron feeding, DMT1 undergoes endocytosis and FPN translocates from the apical cytosol to the basolateral membrane. The fluorescent metallosensor calcein offered to the basolateral surface of enterocytes is found in endosomes in the apical compartment, and its fluorescence is quenched when iron is offered to the apical surface. These experiments are consistent with vesicular iron transport as a possible pathway for intracellular iron transport.     

Transcytosis of epidermal growth factor. The epidermal growth factor receptor mediates uptake but not transcytosis

Madin-Darby canine kidney (MDCK) cells polarize and generate distinct apical and basolateral membrane domains when grown on permeable filter supports. Under these conditions, they transcytose fluid-phase markers. Recently, receptor-mediated transcytosis of epidermal growth factor (EGF) across MDCK cells has been reported (Maratos-Flier, E., Kao, C.-Y. Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595-1601). We examined the role of the EGF receptor in this process. Transcytosis of EGF occurred only in the basolateral-to-apical direction, was time-dependent, and inhibited by the addition of unlabeled EGF in a concentration-dependent manner. In contrast to previous work, we found that only about 5% of basolaterally bound EGF was transported to the apical chamber. The half-time of transport was 90 min. A mutant cell line of MDCK, MDCKII-RCAr, was used to study the expression of the EGF receptor. Cell surface glycoproteins of these mutant cells can be efficiently labeled with [3H]galactose by exogalactosylation. The EGF receptor was found to be expressed only on the basolateral surface. Addition of EGF to the basolateral medium resulted in rapid internalization and degradation of the receptor. Testing directly for transcytosis of basolateral glycoproteins, we detected several proteins transported across the cell. The EGF receptor, however, was not among this group of proteins. Taking these results together, we suggest the following model. Internalization of EGF on the basolateral surface is mediated by the EGF receptor. EGF dissociates from the receptor in an endocytic compartment. A fraction of the EGF is then diverted nonselectively to the transcytotic pathway, as found for other fluid-phase markers previously (Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell Biol. 109, 3243-3258.     

Characterization of the MAL2-positive compartment in oligodendrocytes

Oligodendrocytes (OLs), the myelin-producing cells of the central nervous system, segregate different surface subdomains at the plasma membrane as do other differentiated cells such as polarized epithelia and neurons. To generate the complex membrane system that characterizes myelinating OLs, large amounts of membrane proteins and lipids need to be synthesized and correctly targeted. In polarized epithelia, a considerable fraction of apical proteins are transported by an indirect pathway involving a detour to the basolateral membrane before being internalized and transported across the cell to the apical membrane by a process known as transcytosis. The apical recycling endosome (ARE) or its equivalent, the subapical compartment (SAC), of hepatocytes is an intracellular trafficking station involved in the transcytotic pathway. MAL2, an essential component of the machinery for basolateral-to-apical transcytosis, is an ARE/SAC resident protein. Here, we show that, after differentiation, murine oligodendrocyte precursor and human oligodendroglioma derived cell lines, Oli-neu and HOG, respectively, up-regulate the expression of MAL2 and accumulate it in an intracellular compartment, exhibiting a peri-centrosomal localization. In these oligodendrocytic cell lines, this compartment shares some of the main features of the ARE/SAC, such as colocalization with Rab11a, sensitivity to disruption of the microtubule cytoskeleton with nocodazole, and lack of internalized transferrin. Therefore, we suggest that the MAL2-positive compartment in oligodendrocytic cells could be a structure analogous to the ARE/SAC and might have an important role in the sorting of proteins and lipids for myelin assembly during oligodendrocyte differentiation.     

Differential microtubule requirements for transcytosis in MDCK cells.

Given the role of microtubules in directing the transport of many intracellular organelles, we investigated whether intact microtubules were also required for transcytosis across epithelia. Using polarized MDCK cells expressing receptors for the Fc domain of IgG (FcRII-B2) or polymeric immunoglobulin (pIg-R), we examined the involvement of microtubules in apical to basolateral and basolateral to apical transcytosis, respectively. While depolymerization of microtubules with nocodozole had no effect on apical to basolateral transcytosis via FcR, basolateral to apical transcytosis of dimeric IgA via pIg-R was almost completely blocked. Inhibition due to nocodozole was selective for basolateral to apical transcytosis, since neither endocytosis nor receptor recycling was significantly affected at either plasma membrane domain. As shown by confocal microscopy, the block in transcytosis was due to the inability of MDCK cells to translocate IgA-containing vesicles from the basolateral to the apical cytoplasm in the absence of an intact microtubule network. The nocodazole sensitive step could be partially by-passed, however, by allowing cells to internalize IgA at 17 degrees C prior to nocodazole treatment. Although incubation at 17 degrees C blocked release of IgA into the apical medium, it did not prevent translocation of IgA-containing vesicles to the apical cytoplasm. Thus, receptor-mediated transcytosis in opposite directions exhibits distinct requirements for microtubules, a feature which reflects the spatial organization of MDCK cells.     

Mechanisms of polarized targeting of the FSH receptor]

Gonadotropin and TSH receptors belong to a subgroup of G protein-coupled receptors. TSH and FSH receptor present a particular intracellular traffic: they present a polarized basolateral expression in thyroid follicular cells and in Sertoli cells respectively. By contrast, the LH receptor is expressed circumferentially in target gonadic cells. We expressed these receptors in MDCK cells (a well characterized model of polarized epithelial cells) to understand this difference of properties. We show that the three receptors have a polarized basolateral expression in these cells. All contain a basolateral targeting signal. Furthermore, gonadotropin receptors undergo a partial transcytosis which is not observed for the TSH receptor. We show that heterotrimeric G proteins play a role in this mechanism of transcytosis. This effect is not mediated by adenylate cyclase activation and involves a population of G proteins different from that involved in signal transduction. We thus used in vitro mutagenesis to delineate the basolateral localization signal of the FSH receptor. Surprisingly, the signal is localized in the C-terminal tail of the intracellular domain which is not conserved between the three receptors. It contains 14 amino-acids and its activity is mainly dependent on a tyrosine and a leucine residue. The basolateral localization signal of the FSHR is not colinear with its internalization signal. This signal is autonomous and dominant because, when transferred to an apically targeted membrane protein, the neurotrophin receptor, it redirects the chimeric construct to the basolateral domain of MDCK cells. The basolateral localization signal of the FSH receptor is thus the first signal identified for a G protein-coupled receptor and more generally for a hormone receptor.     

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.     

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.     

Dynamics of MAL2 during glycosylphosphatidylinositol-anchored protein transcytotic transport to the apical surface of hepatoma HepG2 cells

Delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface takes place by transcytosis in hepatocytes and also probably in epithelial Madin-Darby canine cells. The integral protein MAL2 was demonstrated to be essential for basolateral-to-apical transcytosis in hepatoma HepG2 cells. Reduction of endogenous MAL2 levels impedes cargo delivery to the apical membrane, but, paradoxically, cargo does not accumulate in the subapical compartment where MAL2 predominantly resides but in distant endosome elements. To understand how transcytosis can be apparently mediated at a distance, we have analyzed the dynamics of machinery and cargo by live-cell imaging of MAL2 and transcytosing CD59, a GPI-anchored protein, in HepG2 cells. MAL2 was revealed as being a highly dynamic protein. Soon after basolateral endocytosis of CD59, a fraction of MAL2 redistributed into peripheral vesicular clusters that concentrated CD59 and that were accessible to transferrin (Tf) receptor, a basolateral recycling protein. Following Tf receptor segregation, the clusters fused in a MAL2(+)globular structure and moved toward the apical surface for CD59 delivery. All these processes were impaired in cells with reduced MAL2 content. Other GPI-anchored proteins examined behave similarly. As MAL2 is expressed by many types of epithelia, the sorting events described herein are probably of quite general utility.     

Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization

The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA.     

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.     

Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells.

Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N-ethylmaleimide-sensitive factor (NSF)-alpha soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25,000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicle.     

Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked “hyperpolarization,” i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.     

Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.