scholarly journals Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane.

1985 ◽  
Vol 100 (1) ◽  
pp. 27-34 ◽  
Author(s):  
N G Lipsky ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Chinese Hamster ◽  
Fluorescent Labeling ◽  
Lung Fibroblasts ◽  
Cultured Fibroblasts ◽  
Back Exchange ◽  
Pass Through ◽  
Intracellular Translocation

When monolayer cultures of Chinese hamster lung fibroblasts are briefly incubated at 2 degrees C with the fluorescent sphingolipid analogue, C6-NBD-ceramide (N- [7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine), fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occur. During further incubation at 37 degrees C, the Golgi apparatus, and later the plasma membrane, become intensely fluorescent. Within this period, the C6-NBD-ceramide is converted to equal amounts of fluorescent sphingomyelin and glucocerebroside (Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA., 80:2608-2612). In the present study, the intracellular translocation of these metabolites and their subsequent appearance at the plasma membrane were investigated by fluorescence microscopy, the addition of the ionophore monensin, and the technique of "back exchange," in which the amounts and types of fluorescent lipids present at the cell surface are identified after their transfer from the cell surface into recipient vesicles. In control cells, the amount of fluorescent glucocerebroside and sphingomyelin that could be removed from the cell surface by back exchange increased during incubation at 37 degrees C, correlating with the increased fluorescence of the plasma membrane observed by microscopy. In the presence of 10 microM monensin, visible labeling of the plasma membrane was greatly diminished, whereas the Golgi apparatus became highly fluorescent and distended. The ability to remove fluorescent metabolites from the cell surface by back exchange was significantly but reversibly inhibited by monensin. Monensin also increased the total amount of fluorescent sphingomyelin, but not the glucocerebroside found in cells. Subcellular fractions were assayed for their ability to convert radiolabeled and fluorescent ceramides to the corresponding sphingomyelins and glucocerebrosides. The activities of parallel fractions coincided, suggesting that the presence of the NBD moiety did not affect the cellular metabolism of ceramide. Furthermore, the major peak of sphingomyelin- and glucocerebroside-synthesizing activity appeared to coincide with an enriched Golgi fraction. These results strongly suggest that fluorescent sphingomyelin was not synthesized at the plasma membrane as has recently been suggested for endogenous sphingomyelin. Rather, both the sphingomyelin and glucocerebroside analogues were synthesized intracellularly from C6-NBD-ceramide and translocated through the Golgi apparatus to the cell surface.

scholarly journals Truncations of the C-terminal cytoplasmic domain of MG160, a medial Golgi sialoglycoprotein, result in its partial transport to the plasma membrane and filopodia

1998 ◽  
Vol 111 (2) ◽  
pp. 249-260 ◽  
Author(s):  
J.O. Gonatas ◽  
Y.J. Chen ◽  
A. Stieber ◽  
Z. Mourelatos ◽  
N.K. Gonatas
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Type I ◽  
Ovary Cells

MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown. In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia. In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus. This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.

scholarly journals Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus.

1984 ◽  
Vol 99 (2) ◽  
pp. 742-751 ◽  
Author(s):  
R G Sleight ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Lipid Transfer ◽  
Outer Leaflet ◽  
Membrane Lipid Bilayer ◽  
Fluorescent Lipid

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD-PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.

scholarly journals Internalization of Monomeric Lipopolysaccharide Occurs after Transfer Out of Cell Surface Cd14

1999 ◽  
Vol 190 (4) ◽  
pp. 509-522 ◽  
Author(s):  
Thierry Vasselon ◽  
Eric Hailman ◽  
Rolf Thieringer ◽  
Patricia A. Detmers
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Soluble Cd14 ◽  
Stable Transfectants ◽  
Intracellular Vesicles ◽  
Green Fluorescent

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14–EGFP) was used to follow trafficking of mCD14 and BODIPY–LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex–like structure. mCD14–EGFP was functional in mediating binding of and responses to LPS. BODIPY–LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14–EGFP on the cell surface. However, within 5–10 min, the BODIPY–LPS distributed to intracellular vesicles that did not contain mCD14–EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

scholarly journals Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas

1995 ◽  
Vol 310 (1) ◽  
pp. 203-214 ◽  
Author(s):  
C Widmann ◽  
W Dolci ◽  
B Thorens
Keyword(s):  
Plasma Membrane ◽  
Pancreatic Beta Cell ◽  
Chinese Hamster ◽  
Lung Fibroblasts ◽  
Continuous Exposure ◽  
Agonist Binding ◽  
Receptor Endocytosis ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Insulinoma Cells

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

scholarly journals alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

1979 ◽  
Vol 82 (3) ◽  
pp. 614-625 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Coated Vesicles ◽  
The Other ◽  
Con A ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Complexes ◽  
Transmission Electron ◽  
Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2

2003 ◽  
Vol 284 (5) ◽  
pp. C1319-C1329 ◽  
Author(s):  
Dirk Roosterman ◽  
Fabien Schmidlin ◽  
Nigel W. Bunnett
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Calcium Mobilization ◽  
Dominant Negative ◽  
Wild Type ◽  
Early Endosomes ◽  
Protease Activated Receptor 2

We evaluated the contribution of rab5a and rab11a to trafficking and signaling of protease-activated receptor 2 (PAR2), a receptor for trypsin and tryptase. Agonists stimulated internalization of PAR2 into early endosomes containing rab5a. Dominant negative rab5aS34N disrupted early endosomes and inhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 was sorted to lysosomes, and rab5a remained in early endosomes. Rab5a promoted and rab5aS34N impeded resensitization of trypsin-induced calcium mobilization. Rab11a was detected in the Golgi apparatus with PAR2, and PAR2 agonists stimulated redistribution of rab11a into vesicles containing PAR2 that migrated to the cell surface. Dominant negative rab11aS25N was mostly confined to the Golgi apparatus. Although expression of rab11aS25N caused retention of PAR2 in the Golgi apparatus, it did not abolish trafficking of PAR2 to the cell surface. However, expression of wild-type rab11a accelerated both recovery of PAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus to the plasma membrane.

scholarly journals Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation

1997 ◽  
Vol 328 (2) ◽  
pp. 353-361 ◽  
Author(s):  
L. Gergely LUKACS ◽  
Gersana SEGAL ◽  
Norbert KARTNER ◽  
Sergio GRINSTEIN ◽  
Fred ZHANG
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Protein Phosphorylation ◽  
Chinese Hamster ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Hypertonic Medium ◽  
Cystic Fibrosis Transmembrane

Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by > 90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.

scholarly journals Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins.

1987 ◽  
Vol 105 (3) ◽  
pp. 1227-1240 ◽  
Author(s):  
S A Green ◽  
K P Zimmer ◽  
G Griffiths ◽  
I Mellman
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Rat Kidney ◽  
Fc Receptor ◽  
Lysosomal Membrane ◽  
Plasma Membrane Proteins ◽  
Kinetics Of

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.

scholarly journals Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts.

1989 ◽  
Vol 108 (6) ◽  
pp. 2169-2181 ◽  
Author(s):  
M Koval ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Cell Periphery ◽  
Perinuclear Region ◽  
Cultured Fibroblasts ◽  
Plasma Membrane Lipid ◽  
Intracellular Compartments ◽  
Fluorescent Lipid

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.

scholarly journals Ganglioside glycosyltransferases and newly synthesized gangliosides are excluded from detergent-insoluble complexes of Golgi membranes

2004 ◽  
Vol 377 (3) ◽  
pp. 561-568 ◽  
Author(s):  
Pilar M. CRESPO ◽  
Adolfo R. ZURITA ◽  
Claudio G. GIRAUDO ◽  
Hugo J. F. MACCIONI ◽  
Jose L. DANIOTTI
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Chinese Hamster ◽  
Galactosyl Transferase ◽  
Golgi Membranes ◽  
Cell Clones ◽  
Triton X ◽  
Ganglioside Species

GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane GM3, and most GD3 and GT3, reside in GEM. Immunocytochemical examination of GD3-expressing cells showed GD3 to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent GD3, GT3 and GM3 segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.

Sign in / Sign up

Export Citation Format

Share Document