A polarized epithelial cell mutant deficient in translocation of UDP-galactose into the Golgi complex.

102

Abstract. Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of the Golgi complex while enzymes acting in late steps of glycosphingolipid biosynthesis have their active centers in the Golgi lumen. However, the topology of the "early" galactose-transferring enzymes is largely unknown. We used shortchain ceramides with either a 2-hydroxy fatty acid (HFA) or a normal fatty acid (NFA) to determine the topology of the galactosyltransferases involved in the formation of HFA-and NFA-galactosylceramide (GalCer), lactosylceramide (LacCer), and galabiosylceramide (GaECer).Although the HFA-GaICer synthesizing activity colocalized with an E R marker, the other enzyme activities fractionated at the Golgi density of a sucrose gradient. In cell homogenates and permeabilized cells, newly synthesized short-chain GlcCer and GalCer were accessible to serum albumin, whereas LacCer and Ga2Cer were protected. From this and from the results obtained after protease treatment, and after interfering with UDP-Gal import into the Golgi, we conclude that (a) GlcCer and NFA-GaICer are synthesized in the cytosolic leaflet, while LacCer and GaECer are synthesized in the lumenal leaflet of the Golgi. (b) HFA-GalCer is synthesized in the lumenal leaflet of the ER, but has rapid access to the cytosolic leaflet. (c) GlcCer, NFA-GaICer, and HFA-GaICer translocate from the cytosolic to the lumenal leaflet of the Golgi membrane. The transbilayer movement of GlcCer and NFA-GalCer in the Golgi complex is an absolute requirement for higher glycosphingolipid biosynthesis and for the cell surface expression of these monohexosyl sphingolipids.LYCOSPHINGOLIPIDS are universal membrane components of eukaryotic cells. They are enriched at the cell surface and in the lumen of endosomes and lysosomes (60). At the cell surface the glycosphingolipids not only exert functions in cell-cell interaction, cell-substrate interaction, and signal transduction, but are also used as receptors by bacteria, bacterial toxins, and viruses. Glycosphingolipids are a relatively minor constituent of most membranes, but a major component of myelin and of the apical plasma membrane of the polarized epithelial cells that line the gastrointestinal and urinary tract. In the latter tissues, glycosphingolipids play a structural role in rigidifying and protecting the cell surface. The apical plasma membrane of polarized epithelial cells is enriched in glycosphingolipids relative to the basolateral plasma membrane which contains less glycosphingolipids Please address all correspondence to K. Burger, Department of Cell Biology, Universiteit Utrecht, AZU H02.314, 3584 CX Utrecht, The Netherlands, Tel: 31 30 2506480. Fax: 31 30 2541797. E-mail: K.N.J.Burger @med.ruu.nl but more of the phospholipid phosphatidylcholine and the (sphingo)phospholipid sphingomyelin (SM). t Studies using epithelial cells in culture indicate that glycosphingolipids and phospholipids are sorted intracellularly before reaching the cell surface. Moreover, glycosphingolipids are thought to play a key role ...

“…While HepG2 cells contain only trace amounts of GalCer (50), MDCK II cells contain substantial amounts of Gal-Cer and higher derivatives (11,23,39). In subfractionation experiments (Fig.…”

Section: Two Different Ceramide Galactosyltransferase Activities In Mdck H Cellsmentioning

confidence: 93%

Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis.

Burger

Bijl

Meer

1996

102

“…A fraction enriched in Golgi was isolated from a light mitochondrial fraction by flotation on a Nycodenz step gradient (25, 17.5, and 10%), according to the manufacturer's protocol (Centrifugation Techniques VI; Nycomed, Oslo, Norway). Organelle markers (see Results; Table I) were chosen as follows: for mitochondria, succinate dehydrogenase (Centrifugation Techniques IV; Nycomed); for ER, ␣ -glucosidase II fluorescence assay, using 4-methyl umbelliferone (4MU)-␣d -glucoside (Sigma Chemical Co.) as substrate; for Golgi, ␣ -mannosidase II fluorescence assay using 4MU-␣d -mannopyranoside (Sigma Chemical Co.) as substrate (Storrie and Madden, 1990); for TGN, sialyltransferase (Brandli et al, 1988;Simon et al, 1996); for lysosomes, immunoblot analysis of mature form of cathepsin D; for plasma membrane, immunoblot analysis of gp114 or dot blot analysis of biotinylated membranes with 125 I-streptavidin.…”

Section: Vesicle Release From Purified Golgi Fractionsmentioning

confidence: 99%

Myosin II Is Involved in the Production of Constitutive Transport Vesicles from the TGN

Müsch

Cohen

Rodríguez-Boulan

1997

171

145

The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. Here we show that the putative TGN coat protein p200 (Narula, N., I. McMorrow, G. Plopper, J. Doherty, K.S. Matlin, B. Burke, and J.L. Stow. 1992. J. Cell Biol. 114: 1113–1124) is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro, we provide functional evidence that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein (VSVG) from the TGN of polarized MDCK cells. The 50% reduced efficiency in VSVG vesicle release from the TGN in vitro after depletion of p200/myosin II could be reestablished to control levels by the addition of purified nonmuscle myosin II. Several inhibitors of the actin-stimulated ATPase activity of myosin specifically inhibited the release of VSVG-containing vesicles from the TGN.

“…Exogalactosylation of cells was carried out as described (5,12,59) . Cells on polylysine-coated 15-cm tissue culture plates were labeled at 75-90% confluence, usually 1-2 d after plating.…”

Section: Exogalactosylationmentioning

confidence: 99%

Low density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells

Kelly

1992

146

Abstract. Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization . We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a variant clone of PC12, a regulated secretory cell line. The cationindependent mannose 6-phosphate receptor and the low density lipoprotein receptor, both rapidly internalized from the cell surface, and the synaptic vesicle membrane protein synaptophysin, were transported to the Golgi apparatus with equivalent, nonlinear kinetics. Transport to the Golgi apparatus (t 1/2 = 2.5-3.0 h) was several times fäster than turnover of these proteins (ti/2 > 20 h), indicating that transport of these proteins to the Golgi apparatus occurred on average