UMLS:C0038362 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0038362 (stomatitis)
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Immunoisolation techniques have led to the purification of apical and basolateral transport vesicles that mediate the delivery of proteins from the trans-Golgi network to the two plasma membrane domains of MDCK cells. We showed previously that these transport vesicles can be formed and released in the presence of ATP from mechanically perforated cells (Bennett, M. K., A. Wandinger-Ness, and K. Simons, 1988. EMBO (Euro. Mol. Biol. Organ.) J. 7:4075-4085). Using virally infected cells, we have monitored the purification of the trans-Golgi derived vesicles by following influenza hemagglutinin or vesicular stomatitis virus (VSV) G protein as apical and basolateral markers, respectively. Equilibrium density gradient centrifugation revealed that hemagglutinin containing vesicles had a slightly lower density than those containing VSV-G protein, indicating that the two fractions were distinct. Antibodies directed against the cytoplasmically exposed domains of the viral spike glycoproteins permitted the resolution of apical and basolateral vesicle fractions. The immunoisolated vesicles contained a subset of the proteins present in the starting fraction. Many of the proteins were sialylated as expected for proteins existing the trans-Golgi network. The two populations of vesicles contained a number of proteins in common, as well as components which were enriched up to 38-fold in one fraction relative to the other. Among the unique components, a number of transmembrane proteins could be identified using Triton X-114 phase partitioning. This work provides evidence that two distinct classes of vesicles are responsible for apical and basolateral protein delivery. Common protein components are suggested to be involved in vesicle budding and fusion steps, while unique components may be required for specific recognition events such as those involved in protein sorting and vesicle targeting.
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PMID:Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. 220 40

We have developed a highly efficient in vitro-transport assay that couples translocation across the ER membrane and transport to the Golgi complex using the secreted pheromone alpha-factor as a marker protein. Radiolabeled prepro-alpha-factor of high specific radioactivity is obtained by in vitro-translating this protein in a yeast lysate. Prepro-alpha-factor synthesized in vitro is then translocated directly into microsomes or the ER of permeabilized yeast cells. Conversion of the 26-kDa ER form of pro-alpha-factor to the high molecular weight Golgi form is dependent on the presence of ATP and soluble and membrane-bound factors. Differential centrifugation and fractionation on a sucrose gradient have shown that the ER and Golgi forms of alpha-factor are enriched in separate compartments after the transport reaction. These and other findings (see Ruohola et al., 1988, for a more complete discussion) indicate that conversion to the high molecular weight form of alpha-factor is the result of authentic intercompartmental transport. Permeabilized mammalian cells have been used to reconstitute transport from the ER to the Golgi complex. In these systems (Becker et al., 1987; Simons and Virta, 1987), a viral membrane glycoprotein protein (vesicular stomatitis virus G protein) is used as the marker protein. This protein is radiolabeled with [35S]methionine during virus infection, either before or after the cells are permeabilized. Radiolabeled G protein, residing in the ER, is then transported to the Golgi complex in the presence of an ATP-regenerating system. In the mammalian system the donor and acceptor compartments are retained within the permeabilized cells (Simons and Virta, 1987); however, on occasion the addition of an exogenous acceptor compartment is required (Beckers et al., 1987). The assay we developed (Ruohola et al., 1988) differs from the mammalian assay (Beckers et al., 1987) in that we introduce radiolabeled marker protein into the ER in vitro during translocation rather than during virus infection. In addition, in our assay the acceptor Golgi compartment is always provided exogenously to the permeabilized cells. Therefore, if acceptor membranes are present in the PYC, they are not utilized. Because the permeabilized cells and the S3 fraction are prepared differently, the conditions used to prepare the cells may lead to inactivation or loss of the acceptor compartment. The in vitro assay will enable us to purify components involved in transporting proteins from the lumen of the ER to the Golgi complex. Antibody prepared to purified components can be used to clone the genes that code for these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reconstitution of transport from the ER to the Golgi complex in yeast using microsomes and permeabilized yeast cells. 267 24

The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (39.5 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum. After infection with VSV and incubation at 39.5 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (32.5 degrees C). Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle. G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions. The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier. In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (39.5 degrees C) to 18.5 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, K.S., and K. Simons, 1983, Cell, 34:233-243). Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane. When the temperature was subsequently shifted to 32.5 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface. To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces. Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 1.0 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron). Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae.
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PMID:Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses. 298 Dec 29

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.
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PMID:Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells. 299

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 approximately 1.05 g/cm3, 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 degrees 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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PMID:Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical plasma membrane of Madin-Darby canine kidney cells. I. Involvement of endosomes and lysosomes. 608 57

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.
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PMID: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. 608 58

In polarized Madin-Darby canine kidney cells the newly synthesized plasma membrane proteins, on the exocytic pathway, are sorted in the trans-Golgi network (TGN) and delivered directly to the apical or basolateral surface. Forskolin, isobutylmethylxanthine, and dibutyryl cAMP, all known to activate protein kinase A, stimulated transport of influenza hemagglutinin (HA) from the TGN to the apical surface. The same reagents, however, did not affect the transport of HA from the endoplasmic reticulum to the Goli complex nor did they affect transport of vesicular stomatitis virus G protein from the TGN to the basolateral surface. The addition of staurosporin, a general protein kinase inhibitor, did not affect the transport of HA in nontreated cells but blocked the stimulation caused by the above reagents. Apical transport of HA was also stimulated by phorbol ester, an activator of protein kinase C. Activation of apical transport by phorbol ester as well as aluminum fluoride (Pimplikar, S. W., and Simons, K. (1993) Nature 362, 456-458) was also negated by staurosporin. These results show that in polarized Madin-Darby canine kidney cells, protein kinase A and protein kinase C selectively stimulate the apical transport.
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PMID:Activators of protein kinase A stimulate apical but not basolateral transport in epithelial Madin-Darby canine kidney cells. 803 64

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.
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PMID:Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. 860 59

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.
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PMID:Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts. 885 70

Reduction of the cholesterol level in membranes of epithelial Madin-Darby canine kidney (MDCK) cells reverses the apical-to-basolateral transport ratio of the apical membrane marker protein influenza virus haemagglutinin and the secreted glycoprotein gp80. At the same time, basolateral transport of the vesicular stomatitis virus G protein is unaffected [Keller and Simons (1998) J. Cell Biol. 140, 1357-1367]. To investigate whether cholesterol depletion influences apical sorting mechanisms specifically, or apical transport capacity more generally, we studied the effect of cholesterol depletion on the secretion of three different classes of molecules from the apical and basolateral surfaces of MDCK cell layers: glycoprotein gp80, sulphated proteoglycans and proteins, and non-glycosylated rat growth hormone. In each case, cholesterol depletion reduced the fraction secreted to the apical medium and increased the fraction secreted basolaterally. The fact that this was observed for all sulphated proteins and proteoglycans and for the non-glycosylated rat growth hormone, which is randomly secreted in untreated cells, indicates that cholesterol depletion reduces the apical transport capacity, rather than interfering with specific recognition and sorting processes.
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PMID:Cholesterol depletion reduces apical transport capacity in epithelial Madin-Darby canine kidney cells. 1141 30

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