The G protein of vesicular stomatitis virus was implanted in the apical plasma membrane of Madin-Darby canine kidney cells by low pH-dependent fusion of the viral envelope with the cellular membrane. The amount of fusion as determined by removal of unfused virions, either by tryptic digestion or by EDTA treatment at 0°C, was 22-24% of the cell-bound virus radioactivity. Upon incubation of cells after implantation, the amount of G protein as detected by immunofluorescence diminished on the apical membrane and appeared within 30 min on the basolateral membrane. At the same time some G protein fluorescence was also seen in intracellular vacuoles. The observations by immunofluorescence were confirmed and extended by electron microscopy. Using immunoperoxidase localization, G protein was seen to move into irregularly shaped vacuoles (endosomes) and multivesicular bodies and to appear on the basolateral plasma membrane. These results suggest that the apical and basolateral domains of Madin-Darby canine kidney cells are connected by an intracellular route.In most cells the plasma membrane is continuously endocytosed (36) and the loss of surface membrane must be balanced by retrieval of membrane from inside the cell. Membrane recycling poses a special problem in epithelial cells because the plasma membrane is polarized; it is divided into apical and basolateral domains with distinct protein and lipid compositions (35). If the plasma membrane is continuously recycling, an important question is how the apical and basolateral domains preserve their unique compositions. If the apical and the basolateral recycling routes are separated, randomization of the cell surface would of course be prevented. If, however, they connect at some point in the cell, continuous sorting of apical and basolateral proteins would have to take place.In an attempt to map the membrane traffic routes to and from the cell surface in epithelial cells, we are using the MadinDarby canine kidney (MDCK) cell line as our experimental system (7, 16). These cells display both structural and functional polarity when grown in culture (24, 32). The microvillar

The envelope of vesicular stomatitis virus was fused with the apical plasma membrane of Madin-Darby canine kidney cells by low pH treatment. The fate of the implanted G protein was then followed using a protein A-binding assay, which was designed to quantitate the amount of G protein in the apical and the basolateral membranes. The implanted G protein was rapidly internalized at 31 °C, whereas at 10°C no uptake was observed. Already after 15 min at 31°C, a fraction of the G protein could be detected at the basolateral membrane. After 60 min 25-48% of the G protein was basolateral as measured by the protein A-binding assay. At the same time, 25-33% of the implanted G protein was detected at the apical membrane. Internalization of G protein was not affected by 20 mM ammonium chloride or by 10 ~M monensin. However, the endocytosed G protein accumulated in intracellular vacuoles and redistribution back to the plasma membrane was inhibited. We conclude that the implanted G protein was rapidly internalized from the apical surface of Madin-Darby canine kidney cells and a major fraction was routed to the basolateral domain.Enveloped RNA viruses have provided excellent tools to study the intracellular pathway of membrane proteins from their site of synthesis to the plasma membrane (see reference 8). Recently, the use of viruses and their envelope glycoproteins has been extended to study endocytosis of the cell surface (12, 30).In the preceding paper we developed another approach to study the traffic to and from the cell surface in Madin-Darby canine kidney (MDCK) cells (17). In this case the proteins were not introduced into the plasma membrane from within after synthesis, but inserted there from the outside by low pHinduced fusion of the viral envelope with the cellular plasma membrane (16, 3 l, 32). MDCK cells are polarized epithelial cells, the plasma membrane of which is differentiated into two structurally and functionally different domains separated by tight junctions, namely the apical surface facing the growth medium and the basolateral surface facing the neighboring cells and the substratum (4, I l, 19, 20, 24). Normally during vesicular stomatis virus (VSV) infection of MDCK cells the G proteins are mainly transported to the basolateral plasma membrane (25). In the present study we implanted the G protein of VSV into the apical plasma membrane of these MDCK cells. Our previous morphological study (17) showed that the implanted G proteins are rapidly endocytosed and that some of them are distributed to the basolateral surface. In the present study we used a protein A-binding assay to characterize the internalization and redistribution of the implanted G proteins in more detail. MATERIALS AND METHODSThe cells, virus preparations, the implantation procedure of the G protein into the apical plasma membrane of MDCK cells, and the immunofluorescence staining technique are described in our previous study (17). Protein A-binding Assay:The assay was adapted from that described for chicken embryo fibroblasts inf...

The G protein of vesicular stomatitis virus, implanted into the apical plasma membrane of Madin-Darby canine kidney cells, is rapidly transcytosed to the basolateral membrane. In this and the accompanying paper (Pesonen, M., R. Bravo, and K. Simons, 1984, J. Cell Biol. 99:803-809.) we have studied the intracellular route by which the G protein traverses during transcytosis. Using Percoll density gradient centrifugation and free flow electrophoresis we could demonstrate that the G protein is endocytosed into a nonlysosomal compartment with a density of ~1.05 g/cm 3, which has many of the characteristics of endosomes. Transcytosis to the basolateral membrane appeared to occur from this compartment. No direct evidence for the involvement of lysosomes in the transcytotic route could be obtained. No G protein was detected in the lysosomes when transcytosis of G protein was occurring. Moreover, at 21 °C when passage of G protein to the lysosomes was shown to be arrested, transcytosis of G protein could still be demonstrated.

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Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [3H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [3H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins Gl and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)3(GlcNAc)2 peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cistemae in the Golgi region. The relative amounts of [3H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of GI and G2. Endoglycosidase H digestion of isolated [3 5S]methio