Role of Lipid Modifications in Targeting Proteins to Detergent-resistant Membrane Rafts
MANY RAFT PROTEINS ARE ACYLATED, WHILE FEW ARE PRENYLATED*
- From the ‡Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215 and the ¶Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038
Abstract
Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the liquid-ordered phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Madin-Darby canine kidney cell DRMs (other than cytoskeletal contaminants) could be labeled with [3H]palmitate. Association of influenza hemagglutinin with DRMs required all three of its palmitoylated Cys residues. Prenylated proteins, detected by [3H]mevalonate labeling or by blotting for Rap1, Rab5, Gβ, or Ras, were excluded from DRMs. Rab5 and H-Ras each contain more than one lipid group, showing that hydrophobicity alone does not target multiply lipid-modified proteins to DRMs. Partitioning of covalently linked saturated acyl chains into liquid-ordered phase domains is likely to be an important mechanism for targeting proteins to DRMs.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM47897 (to D. A. B.) and GM37547 (to M. G. R.).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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↵§ Present address: Dept. of Biology, Johns Hopkins University, Baltimore, MD 21218.
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↵‖ To whom correspondence should be addressed. Tel.: 516-632-8563; Fax: 516-632-8575; E-mail:dbrown{at}mcbsgi.bio.sunysb.edu.
- Abbreviations:
- DRMs
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detergent-resistant membranes
- MDCK
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Madin-Darby canine kidney
- GPI
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glycosylphosphatidylinositol
- PLAP
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placental alkaline phosphatase
- ECL
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enhanced chemiluminescence
- PAGE
-
polyacrylamide gel electrophoresis
- HA
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influenza hemagglutinin.
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- Received August 18, 1998.
- Revision received November 30, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
