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• W2066887588 abstract "Domains rich in sphingolipids and cholesterol, or rafts, may organize signal transduction complexes at the plasma membrane. Raft lipids are believed to exist in a state similar to the liquid-ordered phase. It has been proposed that proteins with a high affinity for an ordered lipid environment will preferentially partition into rafts (Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999)J. Biol. Chem. 274, 3910–3917). We investigated the possibility that lipid-lipid interactions between lipid-modified proteins and raft lipids mediate targeting of proteins to these domains. G protein monomers or trimers were reconstituted in liposomes, engineered to mimic raft domains. Assay for partitioning of G proteins into rafts was based on Triton X-100 insolubility. Myristoylation and palmitoylation of Gαi were necessary and sufficient for association with liposomes and partitioning into rafts. Strikingly, the amount of fatty-acylated Gαi in rafts was significantly reduced when myristoylated Gαi was thioacylated withcis-unsaturated fatty acids instead of saturated fatty acids such as palmitate. Prenylated βγ subunits were excluded from rafts, whether reconstituted alone or with fatty-acylated α subunits. These results suggest that the structural difference between lipids that modify proteins is one basis for the selectivity of protein targeting to rafts. Domains rich in sphingolipids and cholesterol, or rafts, may organize signal transduction complexes at the plasma membrane. Raft lipids are believed to exist in a state similar to the liquid-ordered phase. It has been proposed that proteins with a high affinity for an ordered lipid environment will preferentially partition into rafts (Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999)J. Biol. Chem. 274, 3910–3917). We investigated the possibility that lipid-lipid interactions between lipid-modified proteins and raft lipids mediate targeting of proteins to these domains. G protein monomers or trimers were reconstituted in liposomes, engineered to mimic raft domains. Assay for partitioning of G proteins into rafts was based on Triton X-100 insolubility. Myristoylation and palmitoylation of Gαi were necessary and sufficient for association with liposomes and partitioning into rafts. Strikingly, the amount of fatty-acylated Gαi in rafts was significantly reduced when myristoylated Gαi was thioacylated withcis-unsaturated fatty acids instead of saturated fatty acids such as palmitate. Prenylated βγ subunits were excluded from rafts, whether reconstituted alone or with fatty-acylated α subunits. These results suggest that the structural difference between lipids that modify proteins is one basis for the selectivity of protein targeting to rafts. liquid-ordered phosphatidylcholine (PC):cholesterol (Chol) liposomes (1:7, mol:mol) phosphatidylcholine:phosphatidylethanolamine:sphingomyelin:cerebroside:cholesterol liposomes (1:1:1:1:2) (16:0–18:0(6–7Dibr)phosphatidylcholine PC:Chol (16:0–18:0(6–7Dibr)phosphatidylcholine SCRL heterotrimeric guanine nucleotide-binding regulatory protein unmodified αi1 subunit of a G protein, MGαi, myristoylated Gαi myristoylated and palmitoylated Gαi farnesylated β1γ2 guanosine 5′-3-O-(thio)triphosphate non-receptor tyrosine kinase detergent-resistant membrane polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Our view of the lateral organization of plasma membrane constituents has evolved in recent years from the conventional picture of membrane proteins diffusing freely in a sea of lipid (1.Singer S.J. Nicolson G.L. Science. 1972; 175: 720-731Crossref PubMed Scopus (6046) Google Scholar). A large body of evidence from studies using cell biological and biophysical approaches suggests that there is selective confinement of lipids and proteins in discrete regions of the membrane (2.Brown R.E. J. Cell Sci. 1998; 111: 1-9Crossref PubMed Google Scholar, 3.Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1719) Google Scholar, 4.Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8052) Google Scholar). These domains, named lipid rafts, are rich in sphingolipids and cholesterol, and appear to be a ubiquitous feature of mammalian cells. Lipid rafts are likely to contribute to the structure and function of caveolae, plasma membrane invaginations that are implicated in a variety of cellular processes, including signal transduction, endocytosis, transcytosis, and cholesterol trafficking. It has been proposed that the spatial concentration of specific sets of proteins increases the efficiency and specificity of signal transduction by facilitating interactions between proteins and by preventing inappropriate cross-talk between pathways. Raft lipids have been proposed to exist in a separate phase from the rest of the bilayer, in a state similar to the liquid-ordered (lo)1 phase described in model membrane (5.Brown D.A. London E. Annu. Rev. Cell. Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2546) Google Scholar, 6.Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (637) Google Scholar). Acyl chains of lipids in the lo phase are tightly packed and highly ordered and extended, similar to those in the gel phase. Thus, lipid structural features (such as saturated acyl chains) that enhance formation of the gel phase can also enhance formation of the lo phase when these lipids are mixed with cholesterol. The presence of unusually long saturated acyl chains on sphingolipids promotes phase separation and formation of the lo phase in mixtures of phospholipids, sphingolipids, and cholesterol at concentrations similar to those in the plasma membrane at 37 °C (7.Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (611) Google Scholar), and is likely to do so in biological membranes as well. Cholesterol- and sphingolipid-rich detergent-resistant membrane (DRMs) can be isolated from mammalian cells (8.Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar). Because there are good correlations between onset of formation of an ordered phase and acquisition of detergent insolubility in model membranes (7.Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (611) Google Scholar, 9.Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), and because DRMs isolated from cells are present in the lophase (10.Ge M. Field K.A. Aneja R. Holowka D. Baird B. Freed J. Biophys. J. 1999; 77: 925-933Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), DRMs are thought to be derived from rafts in living cells. In contrast, cell membranes that are in the conventional disordered phase are fully solubilized by non-ionic detergents. Based on the structural model of rafts described above, it has been postulated that proteins with a high affinity for an ordered lipid environment are selectively recruited to rafts (6.Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (637) Google Scholar). These might be expected to include proteins modified with saturated fatty acyl chains, which could partition favorably into lo phase domains. Indeed, the best characterized DRM targeting signals on proteins are structures that include dual saturated acyl chains. These are glycosylphosphatidylinositol membrane anchors (11.Arreaza G. Brown D.A. J. Biol. Chem. 1995; 270: 23641-23647Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12.Rodgers W. Crise B. Rose J.K. Mol. Cell. Biol. 1994; 14: 5384-5391Crossref PubMed Scopus (226) Google Scholar) (which contain predominantly saturated fatty acids; Ref. 13.McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (799) Google Scholar), modification with tandem amide-linked myristate and thioester-linked palmitate (14.Milligan G. Parenti M. Magee A. Trends Biochem. Sci. 1995; 20: 181-187Abstract Full Text PDF PubMed Scopus (284) Google Scholar, 15.Schenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-364Crossref PubMed Scopus (343) Google Scholar), and modification with tandem thioester-linked palmitate chains (16.Arni S. Keilbaugh S.A. Ostermeyer A.G. Brown D.A. J. Biol. Chem. 1998; 273: 28478-28485Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Palmitoylation is also required for DRM association of the integral membrane proteins LAT (17.Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar) and influenza hemagglutinin (18.Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). The contribution of the lipid modification to raft recruitment is not simply the addition of a hydrophobic moiety, as prenylated proteins are not enriched in DRMs (18.Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). Sphingolipid- and cholesterol-rich liposomes (SCRL), constructed to mimic the lipid composition of DRMs isolated from cells (8.Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar), contain lo phase domains (7.Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (611) Google Scholar) and yield DRMs after detergent extraction (6.Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (637) Google Scholar). SCRL are useful for studying association of purified proteins with DRMs in a defined model system. Purified glycosylphosphatidylinositol-anchored proteins incorporated into SCRL are present in DRMs derived from the liposomes (6.Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (637) Google Scholar, 9.Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), supporting the model that acyl-chain interactions are important in targeting. The heterotrimeric GTP-binding proteins (G proteins) are fatty-acylated and prenylated, and thus are a useful model for examining the role of lipid modifications in targeting proteins to rafts. All G protein α subunits are fatty-acylated with amide-linked myristate, thioester-linked palmitate, or both (19.Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). G protein γ subunits are modified by farnesyl or geranylgeranyl groups (19.Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). In mammalian cells, an unambiguous role for N-myristoylation and prenylation in mediating membrane association of G protein subunits has been established. Gαi family members lackingN-myristate or Gγ subunits lacking prenylation are soluble (19.Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Palmitoylation of G protein α subunits does not appear to be a major determinant of membrane avidity, at least in the presence of Gβγ subunits (20.Morales J. Fishburn S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (81) Google Scholar, 21.Fishburn C.S. Herzmark P. Morales J. Bourne H.R. J. Biol. Chem. 1999; 274: 18793-18800Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 22.Huang C. Duncan J.A. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 412-417Crossref PubMed Scopus (84) Google Scholar), but may play a role in targeting Gα specifically to the plasma membrane (20.Morales J. Fishburn S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (81) Google Scholar, 21.Fishburn C.S. Herzmark P. Morales J. Bourne H.R. J. Biol. Chem. 1999; 274: 18793-18800Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 23.Song J. Dohlman H.G. Biochemistry. 1996; 35: 14806-14817Crossref PubMed Scopus (48) Google Scholar, 24.Dunphy J.D. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), and potentially in targeting proteins to subdomains (25.Song K.S. Sargiacomo M. Galbiati F. Parenti M. Lisanti M.P. Cell. Mol. Biol. 1997; 43: 293-303PubMed Google Scholar, 26.Mumby S.M. Curr. Opin. Cell Biol. 1997; 9: 148-154Crossref PubMed Scopus (238) Google Scholar). Palmitoylation-defective mutants of Gαz, Gαo, and Gpa1p, a G protein α subunit in Saccharomyces cerevisiae, are mislocalized to intracellular membranes (20.Morales J. Fishburn S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (81) Google Scholar, 22.Huang C. Duncan J.A. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 412-417Crossref PubMed Scopus (84) Google Scholar, 23.Song J. Dohlman H.G. Biochemistry. 1996; 35: 14806-14817Crossref PubMed Scopus (48) Google Scholar). However, it is not possible to discern whether mislocalization is attributable to the lack of palmitate or mutation of the palmitoylated cysteine residue. Biochemical and morphological evidence points to the organization of G protein pathways in subdomains of the plasma membrane. All of the components of the hormone-sensitive adenylyl cyclase system appear to be enriched in preparations of low density plasma membrane fragments (27.Huang C. Hepler J.R. Chen L.T. Gilman A.G. Andersone R.G.W. Mumby S.M. Mol. Biol. Cell. 1997; 8: 2365-2378Crossref PubMed Scopus (188) Google Scholar). Gαi and Gβ subunits have a punctate appearance in plasma membrane fragments when detected by immunofluorescence. This punctate distribution of Gαi is corroborated by its clustered appearance when decorated with gold particles in electron micrographs of plasma membrane (22.Huang C. Duncan J.A. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 412-417Crossref PubMed Scopus (84) Google Scholar). Gα subunits are enriched in DRM isolated from cells, but there is disagreement as to the extent of enrichment of Gβγ subunits in these preparations (18.Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 28.Chang W. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. DeGunzburg J. Mumby S. Gilman A. Anderson R. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (311) Google Scholar, 29.Rehm A. Pleogh H.L. J. Cell Biol. 1997; 137: 305-317Crossref PubMed Scopus (28) Google Scholar). Two nonexclusive mechanisms have been proposed for the targeting of Gα subunits to lipid rafts; fatty acylation (25.Song K.S. Sargiacomo M. Galbiati F. Parenti M. Lisanti M.P. Cell. Mol. Biol. 1997; 43: 293-303PubMed Google Scholar, 30.Galbiati F. Volonte D. Meani D. Milligan G. Lublin D.M. Lisanti M.P. Parenti M. J. Biol. Chem. 1999; 274: 5843-5850Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), or interactions with caveolin, a coat protein of caveolae that may function as a scaffolding molecule (31.Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 32.Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar). Most Gαi family members contain the DRM-targeting signal, Met-Gly-Cys, that directsN-myristoylation at Gly-2 and palmitoylation at Cys-3 (19.Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). As with similarly modified nonreceptor tyrosine kinases, mutation of either site in Gαi1 reduces association with DRM isolated from transfected cells (25.Song K.S. Sargiacomo M. Galbiati F. Parenti M. Lisanti M.P. Cell. Mol. Biol. 1997; 43: 293-303PubMed Google Scholar). It is assumed that the failure of the mutant proteins to cofractionate with DRM is due to the loss of the lipid modification. However, this has not been directly demonstrated. A direct protein-protein interaction between Gαi subunits and caveolin has also been proposed as a mechanism for targeting proteins to lipid rafts (33.Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J. Hansen S. Nishimoto I. Lisanti M. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). Caveolin is reported to bind to an amino acid motif in Gαi2, fXfXXXf (where f represents aromatic amino acids Trp, Phe, or Tyr) (32.Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar), and also to interact either directly or indirectly with the amino terminus of Gαi1 (30.Galbiati F. Volonte D. Meani D. Milligan G. Lublin D.M. Lisanti M.P. Parenti M. J. Biol. Chem. 1999; 274: 5843-5850Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The direct binding interaction between purified Gαi and caveolin was not reproduced with purified components (27.Huang C. Hepler J.R. Chen L.T. Gilman A.G. Andersone R.G.W. Mumby S.M. Mol. Biol. Cell. 1997; 8: 2365-2378Crossref PubMed Scopus (188) Google Scholar), raising questions about the nature of the interaction of caveolin with the fXfXXXf motif. Co-immunoprecipitation of Gαi2 with caveolin from transfected cells requires only the NH2-terminal domain of Gαi2, is independent of the fXfXXXf motif, and is dependent upon an intact palmitoylation site (30.Galbiati F. Volonte D. Meani D. Milligan G. Lublin D.M. Lisanti M.P. Parenti M. J. Biol. Chem. 1999; 274: 5843-5850Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The studies published to date do not discriminate whether targeting of proteins to rafts by the Met-Gly-Cys motif is mediated by interactions of the protein's fatty acyl chains with membrane lipids, with other proteins present in domains, or through a mechanism that is dependent upon the primary amino acid sequence of the protein rather than the lipid modification. In this paper, we address the question of whether interactions between the lipid modifications present on G proteins and raft lipids account for their distribution in rafts in cells. The affinity of differentially lipid-modified G proteins for rafts was examined by reconstituting purified subunits into liposomes engineered to mimic rafts. Membrane partitioning was evaluated using susceptibility of the protein to Triton X-100 extraction. We present evidence that the selectivity of protein targeting to rafts relies on the lipid structure that modifies the protein. Phosphatidylcholine (liver), phosphatidylethanolamine (liver), cerebrosides (brain), sphingomyelin (brain), cholesterol (wool grease), and (16:0–18:0 (6–7Dibr)-phosphatidylcholine were obtained from Avanti Polar Lipids Inc. [2-palmitoyl-9,10-3H]Phosphatidylcholine and [3H]palmitate were purchased from NEN Life Science Products. The myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA, stearoyl-CoA, and linoleoyl-CoA lipid derivatives were purchased from Sigma. The detergent Triton X-100 was purchased as an aqueous solution (10%) from Roche Molecular Biochemicals, CHAPS from Calbiochem, and polyoxyethylene 10-lauryl ether from Sigma. The nucleotides guanosine 5′-diphosphate and guanosine 5′-triphosphate were obtained from Sigma. GTPγS was from Roche Molecular Biochemicals. Recombinant nonlipidated Gαi1(Gαi) was expressed and purified from Escherichia coli (34.Linder M.E. Ewald D.A. Miller R.J. Gilman A.G. J. Biol. Chem. 1990; 265: 8243-8251Abstract Full Text PDF PubMed Google Scholar). To obtain myristoylated Gαi1(MGαi), the S. cerevisiae N-myristoyltransferase was expressed together with Gαi in bacteria and MGαipurified as described (35.Mumby S.M. Linder M.E. Methods Enzymol. 1993; 237: 254-268Crossref Scopus (112) Google Scholar). Activity of the different purified Gαi preparations was evaluated by quantification of bound GTPγS (34.Linder M.E. Ewald D.A. Miller R.J. Gilman A.G. J. Biol. Chem. 1990; 265: 8243-8251Abstract Full Text PDF PubMed Google Scholar). Recombinant Gβ1 and hexahistidine-tagged Gγ2 subunits (Gβγ) were produced in Sf9 cells and purified by sequential chromatography with nickel-nitrilotriacetic acid (Qiagen) and Mono Q (Amersham Pharmacia Biotech) as described (36.Kozasa T. Gilman A. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). To evaluate the purity of the preparation, the proteins were resolved by SDS-PAGE and stained with silver nitrate. The procedure was adapted from the protocol of Duncan and Gilman (37.Duncan J.A. Gilman A.G. J. Biol. Chem. 1996; 271: 23594-23600Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Purified recombinant MGαi (4 μm) was incubated in Buffer A (20 mm NaHepes (pH 8.0), 2 mm MgCl2, 1 mm EDTA) containing 7.5 mm CHAPS and 80 μmmyristoyl-CoA (C14:0), palmitoyl-CoA (C16:0), palmitoleoyl-CoA (C16:1), stearoyl-CoA (C18:0) or linoleoyl-CoA (C18:2). For Gαimonomer, MGαi was preincubated for 30 min at 30 °C in the presence of GDP (10 μm) and NaF (10 mm), or GTPγS (10 μm). For experiments with trimeric G proteins, heterotrimer was formed by incubatingMGαi (4 μm) with Gβγ (4 μm) in the presence of GDP (10 μm) for 30 min at 30 °C. The acylation reaction was started with the additions of a mix of the other reagents. The solution was then further incubated for 90 min. All concentrations shown are the final concentrations in the autoacylation reaction. To monitor the efficiency of palmitoylation, GTPγS-boundMGαi (1 μm) was incubated with [3H]palmitoyl-CoA (20 μm, 3300 dpm/pmol) in Buffer A containing 7.5 mm CHAPS. [3H]Palmitoyl-CoA was prepared as detailed by Dunphyet al. (24.Dunphy J.D. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Aliquots of the reaction (10 μl) were removed at different time points and mixed with 30 μl of a solution containing 1% SDS and 2 mg/ml aldolase. Samples were then precipitated at room temperature by the addition of 500 μl of 15% trichloroacetic acid, 2% SDS. Following a 45-min incubation, the samples were filtered on BA85 nitrocellulose filters (Schleicher & Schuell) using a 10-place filter manifold (Hoefer). Each tube was rinsed twice with 4 ml of 6% trichloroacetic acid, 2% SDS. The filters were washed twice with 2 × 2 ml of 6% trichloroacetic acid, 2% SDS, followed by 2 × 2 ml of 6% trichloroacetic acid. The filters were dried, placed in 4 ml of scintillation fluid, and counted by liquid scintillation spectrometry. To monitor the acylation ofMGαi when using acyl-CoAs other than palmitoyl-CoA, an indirect method of labeling was used.MGαi (1 μm) was acylated with 20 μm acyl-CoA (C14:0, C16:0, C16:1, C18:0, C18:2) as described above. The solution was then spiked with [3H]palmitoyl-CoA (20 μm, 6000 dpm/pmol) and the incubation continued for 90 min. The reaction was stopped and quantitated as above. Sequential acylation reactions allowed us to estimate the level of acylation in the first reaction by measuring the inhibition of incorporation of radioactive palmitate. Lipids (6 mg) for making phosphatidylcholine and cholesterol (PC:Chol, 7:1 mol:mol) liposomes or phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, cerebrosides, and cholesterol (SCRL, 1:1:1:1:2) (6.Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (637) Google Scholar) were mixed in a glass screw cap vial, dried under nitrogen gas, lyophilized for 1 h, and stored under argon gas at −20 °C. For the sucrose gradient sedimentation assay, the liposomes were brominated (bromoPC:Chol, bromoSCRL) in order to create a heavier liposome, by replacing half of the phosphatidylcholine with brominated phosphatidylcholine (16:0–18:0(6–7Dibr)-PC). To make [3H]phosphatidylcholine labeled PC:Chol or SCRL, 16 nCi/mg lipids of [2-palmitoyl-9,10-3H]phosphatidylcholine was incorporated in the lipid mixture prior to lyophilization. To prepare liposomes, a vial of dried lipids was first brought to room temperature and lyophilized for 30 min. The lipids were suspended in buffer B (100 mm NaCl, 50 mm NaHepes (pH 8.0), 5 mm MgCl2, 2 mm EDTA) at 2 mg/ml, transferred to a Corning glass screw cap tube, and the tube filled with argon gas. All the buffers used for liposome experiments were bubbled with nitrogen gas for a few minutes to reduce solubilized oxygen. The lipid suspension was sonicated in a bath ultrasonicator (Laboratory Supplies, Inc.) until the solution became partially clear (between 5 and 20 min). The suspension was then frozen and thawed three times by alternative immersion of the tube in baths of methanol/dry ice and water at 30 °C. The liposomes were recovered in the pellet by ultracentrifugation for 30 min at 200,000 × g and suspended in buffer B at 20 mg/ml. Liposomes were discarded after 36 h. Freshly acylated G proteins (40 pmol of M/PGαi orM/PGαiβγ), or mock-acylated Gαi, MGαi, Gβγ, andMGαiβγ were mixed with CHAPS (375 nmol) in a total volume of 12 μl. The protein solution was then mixed with 500 μg of PC:Chol or SCRL, and incubated for 45 min at room temperature. Under those conditions, the molar ratio of CHAPS:phospholipids is 0.72 and only partial solubilization of the liposomes occurs (38.Cladera J. Rigaud J.-L. Villaverde J. Dunach M. Eur. J. Biochem. 1997; 243: 798-804Crossref PubMed Scopus (81) Google Scholar). The mixture was then diluted stepwise at room temperature with buffer B containing either 10 μm GDP in the presence or the absence of 10 mm NaF or 10 μm GTPγS, until the CHAPS concentration reached 2 mm. Equal volumes of buffer were added to the partially solubilized liposomes every 2 min and the solution gently vortexed. To recover the liposomes, the sample was fractionated by centrifugation at 200,000 × g for 30 min at 4 °C and the pellet suspended in nucleotide-supplemented buffer B at 2.2 mg of lipids/ml. A sample of the dilution prior to the fractionation, input (I), and the resulting supernatant (S) and suspended pellet (P) were kept to evaluate the efficiency of G protein incorporation into the liposomes. Quantification of G proteins in the fractions was determined either by GTPγS binding for Gαi-containing samples and/or by image analysis (NIH Image) of the gels resulting from protein resolution by SDS-PAGE and stained with Coomassie Blue. Recoveries of the protein in the supernatant (S) and the suspended pellet (P) following the fractionation averaged more than 90% of the starting material (I), (S+P)/I. The reconstitution efficiencies of G protein was expressed as the percentage of protein associated with the liposome pellet over the total amount of protein recovered, P/(S+P). Sucrose gradients were prepared by freezing (−80 °C) a 15% sucrose solution in buffer B supplemented with 10 μm amounts of the appropriate nucleotide in ultracentrifugation tubes (Beckman), and thereafter allowing the solution to slowly thaw at room temperature. This method gives rise to a linear gradient from ∼5% to 20% sucrose, as determined by the sucrose refractive index measured in the fractions recovered. A sample of reconstituted protein in bromoPC:Chol andbromoSCRL or protein alone in buffer B containing 7.5 mm CHAPS was applied to the top of the gradient and centrifuged at 200,000 × g for 8 h at 4 °C. Fractions were collected and analyzed for total lipid and protein content. Lipids were isolated by chloroform extraction (1:1). Following vigorous mixing, the solution was centrifuged in a tabletop centrifuge and the organic phase was recovered and evaporated under nitrogen gas to ∼10 μl. The concentrated lipid extracts were spotted side by side on a HPTLC plate (Whatman) and allowed to dry. Lipids were revealed by charring (39.Macala L.J., Yu, R.K. Ando S. J. Lipid Res. 1983; 24: 1243-1250Abstract Full Text PDF PubMed Google Scholar). Proteins were detected by immunoblot and visualized by chemiluminescence (Pierce). Reconstituted G proteins were chilled on ice for 10 min. Cold Triton X-100 was added to the suspension at a final concentration of 1% and the preparation incubated on ice for 25 min. The solution was then fractionated by centrifugation at 200,000 × g for 30 min. An equivalent sample from the resulting supernatant and the suspended detergent-resistant fraction were resolved by SDS-PAGE, and proteins were detected by Coomassie Blue staining of the gel. The gel was scanned and the intensity of the bands evaluated by densitometry using NIH Image. The amount of protein in the detergent-resistant fraction was expressed as a percentage of the protein recovered following the fractionation, P/(P+S). MGαi was palmitoylatedin vitro in the presence of GDP and NaF and reconstituted in SCRL as described above. To compare the effect of the activation state of Gαi on its membrane partitioning, NaF and MgCl2 were removed from half of the preparation to return the protein to a GDP-bound inactive state. Accordingly, the solution of reconstituted liposomes was divided, and fractionated by ultracentrifugation. The resulting pellets were washed once in a large volume of buffer B containing 5 μm GDP and 10 mm NaF or" @default.
• W2066887588 created "2016-06-24" @default.
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