Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.

Gottlieb, Theodore; González, Alfonso; Rizzolo, Lawrence J.; Rindler, Michael J.; Adesnik, Milton; Sabatini, David D.

doi:10.1083/jcb.102.4.1242

Cited by 74 publications

(49 citation statements)

References 43 publications

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“…2 We speculate that substratum-dependent GFP-CFTR polarity is attributable to differences in cell differentiation in glassgrown versus filter-grown cells. Significant amounts (up to 50%) of membrane proteins which are distributed in a polarized fashion in cells grown on permeable supports are found on the "opposite" membrane domain in MDCK cells grown on glass coverslips (62,63). Our findings emphasize the need to study trafficking of CFTR and other polarized membrane proteins in physiologically relevant settings.…”

Section: Table II Comparison Of I Sc In Parental and Gfp-cftr Expressmentioning

confidence: 82%

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Moyer¹,

Loffing²,

Schwiebert³

et al. 1998

Journal of Biological Chemistry

152

170

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The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl ؊ ) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl ؊ secretion by stimulating CFTR Cl ؊ channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patchclamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl ؊ secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl ؊ secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

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Section: Table II Comparison Of I Sc In Parental and Gfp-cftr Expressmentioning

confidence: 82%

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Moyer¹,

Loffing²,

Schwiebert³

et al. 1998

Journal of Biological Chemistry

152

170

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“…First, such a block would lead to the loss of VSV G from the cell surface, as has been reported when this step is blocked by chloroquine (Gottlieb et al, 1986); this did not occur in the presence of BFA (see Fig. 4).…”

Section: Endocytosis Of Vsv G and Rh-transferrin Continues In The Prementioning

confidence: 90%

“…VSV G is known to be internalized from the cell surface into early endosomes, where it either recycles to the cell surface or is directed to lysosomes (Gottlieb et al, 1986;Gruenberg and Howell, 1987). When VSV G on the cell surface is crosslinked by antibodies, recycling to the cell surface is abolished and internalized VSV G is targeted to the lysosomal pathway, resulting in a rapid loss of VSV G from the cell surface (Gruenberg and Howell, 1987).…”

Section: Endocytosis Of Vsv G and Rh-transferrin Continues In The Prementioning

confidence: 99%

Post-Golgi membrane traffic: brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling

1992

Trends in Cell Biology

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Abstract. Recent studies using the fungal metabolite brefeldin A (BFA) have provided important insights into the dynamics and the organization of the ER/Golgi membrane system. Here we examined the effect of BFA on the functional integrity of the distal part of the secretory pathway, i.e., transport between transGolgi cisternae and the cell surface. To assay export via the constitutive pathway, we followed the movement of vesicular stomatitis virus (VSV) G glycoprotein that had been accumulated in the trans-Golgi network (TGN) by incubation of infected BHK-21 cells at 20~ Addition of BFA rapidly and reversibly inhibited cell surface transport of G protein. The block to secretion was not due to redistribution of externalized G protein to internal pools. It was also not due to collapse of TGN to the ER, since VSV G protein blocked in treated cells resided in compartments that were distinct from the ER/Golgi system. Similar effects were found with a bulk-flow marker: BFA blocked constitutive secretion of glycosaminoglycan chains that had been synthesized and sulfated in the trans-Golgi cisternae. To examine export via the regulated secretory pathway, we assayed secretion of [35S]SO4 labeled secretogranin II from PC12 cells, a marker that has been used to study secretory granule budding from the TGN (Tooze, S. A., U. Weiss, and W. B. Huttner. 1990. Nature [Lond.]. 347:207-208). BFA potently inhibited secretion of sulfated secretogranin II induced by K + depolarization. Inhibition was at the level of granule formation, since BFA had no effect on regulated secretion from preformed granules. Taken together, the results suggest that BFA blocks export via both the constitutive and the regulated pathways. In contrast, endocytosis and recycling of VSV G protein were not blocked by BFA, consistent with previous studies that endocytosis is unaffected (Misumi, Y., Y.

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“…Sections were either cut at 70 nm and stained with uranyl acetate and lead citrate or cut up to 1-m thick, stabilized with a thin film of evaporated carbon, and viewed unstained, in a Philips CM12 electron microscope. To increase the detection of APP-TR chimeras morphologically, monolayers were incubated in 10 mM sodium butyrate 12-16 h at 37°C prior to experimentation (35). Identical results were obtained in experiments performed without the inclusion of sodium butyrate.…”

Section: Pulse-chase Analysis Of Degradation Of App-tr Chimeras In MDmentioning

confidence: 99%

Signal-dependent Trafficking of β-Amyloid Precursor Protein-Transferrin Receptor Chimeras in Madin-Darby Canine Kidney Cells

Lai¹,

Gibson²,

Hopkins³

et al. 1998

Journal of Biological Chemistry

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We have investigated the intracellular trafficking of a chimeric molecule consisting of the cytoplasmic domain of the ␤-amyloid precursor protein (APP) and the transmembrane region and external domain of the human transferrin receptor (TR) in Madin-Darby canine kidney cells. Newly synthesized APP-TR chimeras are selectively targeted to the basolateral surface by a tyrosinedependent sorting signal in the APP cytoplasmic tail. APP-TR chimeras are then rapidly internalized from the basolateral surface and a significant fraction (ϳ20 -30%) are degraded. Morphological studies show that APP-TR chimeras internalized from the basolateral surface are found in tubulo-vesicular endosomal elements, internal membranes of multivesicular bodies, and lysosomes. APP-TR chimeras are also found in 60-nm diameter vesicles previously shown to selectively deliver wild-type TR to the basolateral surface; this result is consistent with the fact that 90% of internalized chimeras that are not degraded are selectively recycled back to the basolateral surface. APP-TR chimeras internalized from the apical surface are selectively transcytosed to the basolateral surface underscoring the importance of basolateral sorting in the endocytic pathway for maintaining the polarized phenotype. Tyr-653, an important element of the YTSI internalization signal in the APP cytoplasmic domain, is required for basolateral sorting in the biosynthetic and endocytic pathways. However, the structural features for basolateral sorting differ from those required for internalization. Alzheimer's disease (AD)1 is a progressive neurodegenerative disorder affecting ϳ1-6% of people over the age of 65. A characteristic neuropathological feature of AD is the senile plaque, which contains ␤-amyloid (A␤), a 39 -43 amino acid peptide derived from the ␤-amyloid precursor protein (APP), a type I integral transmembrane protein whose cellular function is unknown (1, 2). Located on chromosome 21, the gene encoding APP is alternatively-spliced, and several isoforms of differing amino acid lengths are expressed. Genetic studies have revealed mutations in the coding region of APP genes isolated from a small minority of affected individuals with early onset familial AD, and it has been shown that one of these mutations, the Swedish double mutation, leads to secretion of elevated levels of A␤ (3, 4). These observations led to the hypothesis that increased or abnormal A␤ production plays a central role in the pathogenesis of AD. While it is clear that generation of A␤ necessarily involves two proteolytic cleavages of APP: one in the extracellular domain and another in the transmembrane region, how APP is converted to A␤ in terms of proteolytic enzymes involved, cellular location of these cleavage events, and factors regulating its production remain to be determined (2). An alternative proteolytic cleavage by another unidentified enzyme (referred to as ␣-secretase) thought to reside on or near the plasma membrane leads to production of a large soluble fragment (APP s ) comprised of most...

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Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.

Abstract: Abstract. Previous studies (Rindler, M. J.,

Cited by 74 publications

References 43 publications

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Post-Golgi membrane traffic: brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling

Signal-dependent Trafficking of β-Amyloid Precursor Protein-Transferrin Receptor Chimeras in Madin-Darby Canine Kidney Cells

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