scholarly journals Individual Palmitoyl Residues Serve Distinct Roles in H-Ras Trafficking, Microlocalization, and Signaling

2005 ◽  
Vol 25 (15) ◽  
pp. 6722-6733 ◽  
Author(s):  
Sandrine Roy ◽  
Sarah Plowman ◽  
Barak Rotblat ◽  
Ian A. Prior ◽  
Cornelia Muncke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Lipid Rafts ◽  
Lipid Bilayer ◽  
Spatial Organization ◽  
Cysteine Residues ◽  
Wild Type ◽  
Membrane Affinity ◽  
Lateral Segregation

ABSTRACT H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

scholarly journals Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake

2013 ◽  
Vol 305 (7) ◽  
pp. G513-G519 ◽  
Author(s):  
Umadevi Ramasamy ◽  
M. Sawkat Anwer ◽  
Christopher M. Schonhoff
Keyword(s):  
Nitric Oxide ◽  
Plasma Membrane ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Wild Type ◽  
Reactive Compound

The Na+taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.

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 Motility and ultrastructure of large granular lymphocytes on lipid bilayers reconstituted with adhesion receptors LFA-1, ICAM-1, and two isoforms of LFA-3.

1991 ◽  
Vol 115 (3) ◽  
pp. 861-871 ◽  
Author(s):  
O Carpén ◽  
M L Dustin ◽  
T A Springer ◽  
J A Swafford ◽  
L A Beckett ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Lower Percentage ◽  
Target Cells ◽  
Complex Stimulus ◽  
Lymphocyte Function ◽  
Large Granular Lymphocytes ◽  
Granular Lymphocytes

Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here.

Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism

2013 ◽  
Vol 454 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Anna Frick ◽  
Michael Järvå ◽  
Mikael Ekvall ◽  
Povilas Uzdavinys ◽  
Maria Nyblom ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Water Transport ◽  
Water Permeability ◽  
Environmental Changes ◽  
Spinacia Oleracea ◽  
Cysteine Residues ◽  
Plasma Membrane Intrinsic Protein ◽  
Plant Plasma Membrane

Water transport across cellular membranes is mediated by a family of membrane proteins known as AQPs (aquaporins). AQPs were first discovered on the basis of their ability to be inhibited by mercurial compounds, an experiment which has followed the AQP field ever since. Although mercury inhibition is most common, many AQPs are mercury insensitive. In plants, regulation of AQPs is important in order to cope with environmental changes. Plant plasma membrane AQPs are known to be gated by phosphorylation, pH and Ca2+. We have previously solved the structure of the spinach AQP SoPIP2;1 (Spinacia oleracea plasma membrane intrinsic protein 2;1) in closed and open conformations and proposed a mechanism for how this gating can be achieved. To study the effect of mercury on SoPIP2;1 we solved the structure of the SoPIP2;1–mercury complex and characterized the water transport ability using proteoliposomes. The structure revealed mercury binding to three out of four cysteine residues. In contrast to what is normally seen for AQPs, mercury increased the water transport rate of SoPIP2;1, an effect which could not be attributed to any of the cysteine residues. This indicates that other factors might influence the effect of mercury on SoPIP2;1, one of which could be the properties of the lipid bilayer.

Mammalian GPI-anchor modifications and the enzymes involved

2020 ◽  
Vol 48 (3) ◽  
pp. 1129-1138 ◽  
Author(s):  
Yi-Shi Liu ◽  
Morihisa Fujita
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Mammalian Cells ◽  
Gpi Anchor ◽  
C Terminus ◽  
Lipid Moiety ◽  
Human Proteins

Glycosylphosphatidylinositol (GPI) is a glycolipid added to the C-terminus of a large variety of proteins in eukaryotes, thereby anchoring these proteins to the cell surface. More than 150 different human proteins are modified with GPI, and GPI-anchored proteins (GPI-APs) play critical roles in embryogenesis, neurogenesis, immunity, and fertilization. GPI-APs are biosynthesized in the endoplasmic reticulum (ER) and transported to the plasma membrane via the Golgi apparatus. During transport, GPI-APs undergo structural remodeling that is important for the efficient folding and sorting of GPI-APs. Asparagine-linked glycan-dependent folding and deacylation by PGAP1 work together to ensure that correctly folded GPI-APs are transported from the ER to the Golgi. Remodeling of the GPI lipid moiety is critical for the association of GPI-APs with lipid rafts. On the cell surface, certain GPI-APs are cleaved by GPI cleavage enzymes and released from the membrane, a key event in processes such as spermatogenesis and neurogenesis. In this review, we discuss the enzymes involved in GPI-AP biosynthesis and the fate of GPI-APs in mammalian cells, with a focus on the assembly, folding, degradation, and cleavage of GPI-APs.

scholarly journals Caveolae regulate the nanoscale organization of the plasma membrane to remotely control Ras signaling

2014 ◽  
Vol 204 (5) ◽  
pp. 777-792 ◽  
Author(s):  
Nicholas Ariotti ◽  
Manuel A. Fernández-Rojo ◽  
Yong Zhou ◽  
Michelle M. Hill ◽  
Travis L. Rodkey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Molecular Mechanisms ◽  
Gene Array ◽  
Ras Signaling ◽  
Cellular Lipid ◽  
Caveolin 1 ◽  
Signal Output ◽  
Lateral Segregation ◽  
Array Analyses

The molecular mechanisms whereby caveolae exert control over cellular signaling have to date remained elusive. We have therefore explored the role caveolae play in modulating Ras signaling. Lipidomic and gene array analyses revealed that caveolin-1 (CAV1) deficiency results in altered cellular lipid composition, and plasma membrane (PM) phosphatidylserine distribution. These changes correlated with increased K-Ras expression and extensive isoform-specific perturbation of Ras spatial organization: in CAV1-deficient cells K-RasG12V nanoclustering and MAPK activation were enhanced, whereas GTP-dependent lateral segregation of H-Ras was abolished resulting in compromised signal output from H-RasG12V nanoclusters. These changes in Ras nanoclustering were phenocopied by the down-regulation of Cavin1, another crucial caveolar structural component, and by acute loss of caveolae in response to increased osmotic pressure. Thus, we postulate that caveolae remotely regulate Ras nanoclustering and signal transduction by controlling PM organization. Similarly, caveolae transduce mechanical stress into PM lipid alterations that, in turn, modulate Ras PM organization.

scholarly journals Interferon-γ inhibits enterocyte migration by reversibly displacing connexin43 from lipid rafts

2008 ◽  
Vol 295 (3) ◽  
pp. G559-G569 ◽  
Author(s):  
Cynthia L. Leaphart ◽  
Shipan Dai ◽  
Steven C. Gribar ◽  
Ward Richardson ◽  
John Ozolek ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Gap Junctions ◽  
Lipid Rafts ◽  
Interferon Γ ◽  
Dependent Manner ◽  
Wild Type ◽  
Kinase C ◽  
Intercellular Channels ◽  
Ifn Treatment

Necrotizing enterocolitis (NEC) is associated with the release of interferon-γ (IFN) by enterocytes and delayed intestinal restitution. Our laboratory has recently demonstrated that IFN inhibits enterocyte migration by impairing enterocyte gap junctions, intercellular channels that are composed of connexin43 (Cx43) monomers and that are required for enterocyte migration to occur. The mechanisms by which IFN inhibits gap junctions are incompletely understood. Lipid rafts are cholesterol-sphingolipid-rich microdomains of the plasma membrane that play a central role in the trafficking and signaling of various proteins. We now hypothesize that Cx43 is present on enterocyte lipid rafts and that IFN inhibits enterocyte migration by displacing Cx43 from lipid rafts in enterocytes. We now confirm our previous observations that intestinal restitution is impaired in NEC and demonstrate that Cx43 is present on lipid rafts in IEC-6 enterocytes. We show that lipid rafts are required for enterocyte migration, that IFN displaces Cx43 from lipid rafts, and that the phorbol ester phorbol 12-myristate 13-acetate (PMA) restores Cx43 to lipid rafts after treatment with IFN in a protein kinase C-dependent manner. IFN also reversibly decreased the phosphorylation of Cx43 on lipid rafts, which was restored by PMA. Strikingly, restoration of Cx43 to lipid rafts by PMA or by transfection of enterocytes with adenoviruses expressing wild-type Cx43 but not mutant Cx43 is associated with the restoration of enterocyte migration after IFN treatment. Taken together, these findings suggest an important role for lipid raft-Cx43 interactions in the regulation of enterocyte migration during exposure to IFN, such as NEC.

scholarly journals O-Glycosylation as a Sorting Determinant for Cell Surface Delivery in Yeast

2004 ◽  
Vol 15 (4) ◽  
pp. 1533-1543 ◽  
Author(s):  
Tomasz J. Proszynski ◽  
Kai Simons ◽  
Michel Bagnat
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Membrane Protein ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Chimeric Protein ◽  
Initial Step ◽  
Integral Membrane Protein ◽  
Wild Type

Little is known about the mechanisms that determine localization of proteins to the plasma membrane in Saccharomyces cerevisiae. The length of the transmembrane domains and association of proteins with lipid rafts have been proposed to play a role in sorting to the cell surface. Here, we report that Fus1p, an O-glycosylated integral membrane protein involved in cell fusion during yeast mating, requires O-glycosylation for cell surface delivery. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A chimeric protein lacking O-glycosylation motif was missorted to the vacuole and accumulated in late Golgi in wild-type cells. Exocytosis of this protein could be restored by addition of a 33-amino acid portion of an O-glycosylated sequence from Fus1p. Our data suggest that O-glycosylation functions as a sorting determinant for cell surface delivery of Fus1p.

scholarly journals Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

2016 ◽  
Vol 27 (13) ◽  
pp. 2090-2106 ◽  
Author(s):  
Adriana M. Zimnicka ◽  
Yawer S. Husain ◽  
Ayesha N. Shajahan ◽  
Maria Sverdlov ◽  
Oleg Chaga ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Conformational Change ◽  
Structural Component ◽  
Spatial Organization ◽  
Simultaneous Increase ◽  
Wild Type ◽  
Western Blots ◽  
Caveolin 1 ◽  
Spinning Disk ◽  
Fret Efficiency

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or “spreading” of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

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