Lipid-dependent Targeting of G Proteins into Rafts*
- From the ‡Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the §Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794-5215
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.
Footnotes
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↵* This work was supported by a postdoctoral fellowship from the Missouri affiliate of the American Heart Association (to S. M.) and National Institutes of Health Grants GM51466 (to M. E. L.) and GM47897 (to D. A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8228, St. Louis, MO 63110. Tel.: 314-362-6040; Fax: 314-362-7463; E-mail: mlinder@cellbio.wustl.edu.
- Abbreviations:
- lo
-
liquid-ordered
- PC:Chol
-
phosphatidylcholine (PC):cholesterol (Chol) liposomes (1:7, mol:mol)
- SCRL
-
phosphatidylcholine:phosphatidylethanolamine:sphingomyelin:cerebroside:cholesterol liposomes (1:1:1:1:2)
- bromoPC:Chol
-
(16:0–18:0(6–7Dibr)phosphatidylcholine PC:Chol
- bromoSCRL
-
(16:0–18:0(6–7Dibr)phosphatidylcholine SCRL
- G proteins
-
heterotrimeric guanine nucleotide-binding regulatory protein
- Gαi
-
unmodified αi1 subunit of a G protein, MGαi, myristoylated Gαi
- M/PGαi
-
myristoylated and palmitoylated Gαi
- Gβγ
-
farnesylated β1γ2
- GTPγS
-
guanosine 5′-3-O-(thio)triphosphate
- NRTK
-
non-receptor tyrosine kinase
- DRM
-
detergent-resistant membrane
- PAGE
-
polyacrylamide gel electrophoresis
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
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- Received September 27, 1999.
- Revision received November 2, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
