The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo.
(25/52017)
Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules. (+info)
Formation of lipid-linked sugar compounds in Halobacterium salinarium. Presumed intermediates in glycoprotein synthesis.
(26/52017)
The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity. (+info)
Lipolytic action of cholera toxin on fat cells. Re-examination of the concept implicating GM1 ganglioside as the native membrane receptor.
(27/52017)
The possible role of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1) ganglioside in the lipolytic activity of cholera toxin on isolated fat cells has been examined. Analyses of the ganglioside content and composition of intact fat cells, their membranous ghosts, and the total particulate fraction of these cells indicate that N-acetylneuraminylgalactosylglucosylceramide (GM3) represents the major ganglioside, with substantial amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2) and smaller amounts of other higher homologues also present. Native GM1 was not detected in any of these preparations. Examination of the relative capacities of various exogenously added radiolabeled sphingolipids to bind to the cells indicated that GM2 and glucosylsphingosine were accumulated by the cells to extents comparable to GM1. Galactosylsphingosine and sulfatide also exhibited significant, although lesser, binding affinities for the cells. The adipocytes appeared to nonspecifically bind exogenously added GM1; saturation of binding sites for GM1 could not be observed up to the highest concentration tested (2 X 10(-4) M), wherein about 7 X 10(9) molecules were associated with the cells. Essentially all of this exogenously added GM1 was found bound to the plasma membrane "ghost" fraction. Investigation of the biological responses of the cells confirmed their sensitivities to both cholera toxin and epinephrine-stimulated lipolysis, as well as the lag period displayed during the toxin's action. While we could confirm that the toxin's lipolytic activity can be enhanced by prior treatment of the fat cells with GM1, several of the observed characteristics of this phenomenon differ from earlier reported findings. Accordingly, added GM1 was able to enhance only the subsequent rate, but not the extent, of toxin-stimulated glycerol release (lipolysis) from the cells. We also were unable to confirm the ability of GM1 to enhance the toxin's activity at either saturating or at low toxin concentrations. The limited ability of added GM1 to enhance the toxin's activity appeared in a unique bell-shaped dose-response manner. The inability of high levels of GM1 to stimulate a dose of toxin that was ineffective on native cells suggests that the earlier reported ability of crude brain gangliosides to accomplish this was due to some component other than GM1 in the crude extract. While several glycosphingolipids and some other carbohydrate-containing substances that were tested lacked the ability to mimic the enhancing effect of GM1, 4-methylumbelliferyl-beta-D-galactoside exhibited an effect similar to, although less pronounced than, that of GM1. The findings in these studies are unable to lend support to the earlier hypothesis that (a) GM1 is cholera toxin's naturally occurring membrane receptor on native fat cells, and (b) the ability of exogenously added GM1 to enhance the toxin's lipolytic activity represents the specific creation of additional natural receptors on adipocytes... (+info)
Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin.
(28/52017)
Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance. (+info)
Participation of a trisaccharide-lipid in glycosylation of oviduct membrane glycoproteins.
(29/52017)
Preincubation of a hen oviduct membrane preparation with UDP-Nactyl[14C]glucosamine and bacitracin, followed by incubation with GDP-mannose, leads to formation of a chloroform/methanol (2/1)-extractable glycolipid. Treatment of the lipid with mild acid results in the release of a trisaccharide shown to have the structure beta-mannosyl-N-acetylglucosamineyl-N-acetylglucosamine. Incubation of purified trisaccharide-lipid with oviduct membranes in the presence of sodium deoxycholate, Mn2+, and GDP-mannose leads to formation of a labeled glycoprotein with an apparent molecular weight of 25,000... (+info)
Perturbation of mammalian cell division. III. The topography and kinetics of extrusion subdivision.
(30/52017)
If mitotic-arrested, cold-stored HeLa cells are incubated at 37 degrees C a proportion of the population divides by an aberrant process which we have called subdivision by extrusion. This process has been studied by time-lapse photography and shown to differ from normal cleavage in several respects. The cell surface becomes more generally mobile and, instead of producing the precisely localized furrowing activity of cytokinesis, gives rise to multiple surface protrusions. These protrusions enlarge at the expense of the parent cell and develop into a cluster of small daughter cells (mini segregants). The surface structure of the cell, as seen by scanning electron microscopy, also changes; the microvilli characteristic of interphase, metaphase and cleaving HeLa cells are lost during extrusion and the cell surface becomes smooth. Extrusion activity is much more variable than division by cleavage in terms of both topography and kinetics, and in general takes longer to complete. Some cells in the cold-treated populations divide by mixtures of cleavage and extrusion or by cleavage alone. The relative numbers of cells dividing in different ways vary with the conditions of pretreatment and incubation of the mitotic cells. The greater the perturbation (e.g. longer cold storage), the greater the proportion of extruding rather than cleaving cells. Human diploid cells can also be induced to subdivide by extrusion. Possible mechanisms underlying the different types of division activity are discussed. (+info)
The preprophase band: possible involvement in the formation of the cell wall.
(31/52017)
Numerous vesicles were observed among the microtubules of the "preprophase" band in prophase cells from root tips of Allium cepa. The content of these vesicles looks similar to the matrix of adjacent cell walls, and these vesicles often appear to be involved in exocytosis. In addition, the cell walls perpendicular to the plane of (beneath) the preprophase band are often differentially thickened compared to the walls lying parallel to the plane of the band. Our interpretation of these observations is that the preprophase band may direct or channel vesicles containing precursors of the cell wall to localized regions of wall synthesis. The incorporation of constituents of the cell wall into a narrow region defined by the position of the preprophase band may be a mechanism that ensures unidirecitonal growth of meristematic cells. (+info)
Freeze-fracture studies of frog atrial fibres.
(32/52017)
The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the "gap junction" or "nexus". The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones. (+info)