Antibody binding to glycolipids and glycophosphatidylinositol (GP1)-anchored proteins of lymphocy... more Antibody binding to glycolipids and glycophosphatidylinositol (GP1)-anchored proteins of lymphocytes can trigger activation of specific signal transduction pathways. The finding that GPI-anchored proteins are present in detergent-insoluble complexes with several tyrosine kinases of the Src family suggested that these complexes may represent membrane microdomains involved in the transduction of signals to the cell interior. Recent work has suggested a link between detergentinsoluble microdomains and plasma membrane invaginations termed caveolae. Here we show that lymphocytes lack plasma membrane domains with the characteristic features of caveolae. Furthermore, VIP21caveolin was not detectable in four different lymphocyte cell lines at the protein or mRNAlevel. In addition to the lack of caveolar domains, capping experiments suggested that the bulk of the GPI-anchored protein Thyl and the glycosphingolipid GMl were not stably associated in the lymphocyte plasma membrane. Despite this, Thyl and GMI were present in detergent-insoluble complexes. W e conclude that detergent insolubility does not correlate with the presence of caveolae or of VIP21caveolin and that caveolae, as defined by a number of different markers, are not involved in signal transduction in lymphocytes. Cross-linking of glycophosphatidylinositol (GP1)'-anchored surface glycoproteins can trigger the activation of specific signal transduction pathways in lymphocytes (for review see Ref. 1). As GPI-anchored proteins are anchored in the outer leaflet of the lipid bilayer, the mechanism by which such signals are transmitted across the bilayer to the cytoplasmic surface is not clear. The finding that GPI-anchored proteins are present in insoluble complexes with tyrosine kinases of the Src family (2-5) suggested the existence of membrane microdomains that Mobility Programme (to A. M. F.
The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell l... more The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, NMDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20°C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37°C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20°C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing.
A number of in vitro and in vivo studies have shown that the large superfamily of Ras-related low... more A number of in vitro and in vivo studies have shown that the large superfamily of Ras-related low molecular weight GTP-binding proteins includes proteins which are involved in the control of membrane traffic. In the yeast Saccharomyces cerevisiae, the SEC4 gene encodes a 23 Kd ras-related GTP-binding protein involved in the regulation of vesicular traffic from the Golgi apparatus to the plasma membrane. The protein is found associated with both the cytoplasmic surface of the plasma membrane and secretory vesicles (Salminen and Novick, 1987; Goud et al., 1988). Temperature-sensitive and dominant SEC4 mutants lead to a block in transport from the Golgi apparatus and accumulation of post-Golgi secretory vesicles (Salminen and Novick, 1987; Walworth et al., 1989). The YPT1 gene product is a 23 Kd GTP-binding protein (Gallwitz et al., 1983) which functions at an earlier step of the secretory pathway, from the ER to or within the Golgi apparatus (Schmitt et al., 1986; 1988; Segev and Botstein, 1987; Segev et al., 1988; Baker et al, 1990).
Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or ... more Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or interact with the lipids on either side of the two leaflets. Although recent advances in lipid analytics show that membranes in eukaryotic cells contain hundreds of different lipid species, the function of this lipid diversity remains enigmatic. The basic structure of cell membranes is the lipid bilayer, composed of two apposing leaflets, forming a twodimensional liquid with fascinating properties designed to perform the functions cells require. To coordinate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. This principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to focus and regulate membrane bioactivity. Here we will review the emerging principles of membrane architecture with special emphasis on lipid organization and domain formation.
In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influe... more In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an aDcal plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells
Antibody binding to glycolipids and glycophosphatidylinositol (GP1)-anchored proteins of lymphocy... more Antibody binding to glycolipids and glycophosphatidylinositol (GP1)-anchored proteins of lymphocytes can trigger activation of specific signal transduction pathways. The finding that GPI-anchored proteins are present in detergent-insoluble complexes with several tyrosine kinases of the Src family suggested that these complexes may represent membrane microdomains involved in the transduction of signals to the cell interior. Recent work has suggested a link between detergentinsoluble microdomains and plasma membrane invaginations termed caveolae. Here we show that lymphocytes lack plasma membrane domains with the characteristic features of caveolae. Furthermore, VIP21caveolin was not detectable in four different lymphocyte cell lines at the protein or mRNAlevel. In addition to the lack of caveolar domains, capping experiments suggested that the bulk of the GPI-anchored protein Thyl and the glycosphingolipid GMl were not stably associated in the lymphocyte plasma membrane. Despite this, Thyl and GMI were present in detergent-insoluble complexes. W e conclude that detergent insolubility does not correlate with the presence of caveolae or of VIP21caveolin and that caveolae, as defined by a number of different markers, are not involved in signal transduction in lymphocytes. Cross-linking of glycophosphatidylinositol (GP1)'-anchored surface glycoproteins can trigger the activation of specific signal transduction pathways in lymphocytes (for review see Ref. 1). As GPI-anchored proteins are anchored in the outer leaflet of the lipid bilayer, the mechanism by which such signals are transmitted across the bilayer to the cytoplasmic surface is not clear. The finding that GPI-anchored proteins are present in insoluble complexes with tyrosine kinases of the Src family (2-5) suggested the existence of membrane microdomains that Mobility Programme (to A. M. F.
The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell l... more The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, NMDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20°C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37°C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20°C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing.
A number of in vitro and in vivo studies have shown that the large superfamily of Ras-related low... more A number of in vitro and in vivo studies have shown that the large superfamily of Ras-related low molecular weight GTP-binding proteins includes proteins which are involved in the control of membrane traffic. In the yeast Saccharomyces cerevisiae, the SEC4 gene encodes a 23 Kd ras-related GTP-binding protein involved in the regulation of vesicular traffic from the Golgi apparatus to the plasma membrane. The protein is found associated with both the cytoplasmic surface of the plasma membrane and secretory vesicles (Salminen and Novick, 1987; Goud et al., 1988). Temperature-sensitive and dominant SEC4 mutants lead to a block in transport from the Golgi apparatus and accumulation of post-Golgi secretory vesicles (Salminen and Novick, 1987; Walworth et al., 1989). The YPT1 gene product is a 23 Kd GTP-binding protein (Gallwitz et al., 1983) which functions at an earlier step of the secretory pathway, from the ER to or within the Golgi apparatus (Schmitt et al., 1986; 1988; Segev and Botstein, 1987; Segev et al., 1988; Baker et al, 1990).
Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or ... more Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or interact with the lipids on either side of the two leaflets. Although recent advances in lipid analytics show that membranes in eukaryotic cells contain hundreds of different lipid species, the function of this lipid diversity remains enigmatic. The basic structure of cell membranes is the lipid bilayer, composed of two apposing leaflets, forming a twodimensional liquid with fascinating properties designed to perform the functions cells require. To coordinate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. This principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to focus and regulate membrane bioactivity. Here we will review the emerging principles of membrane architecture with special emphasis on lipid organization and domain formation.
In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influe... more In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an aDcal plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells
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