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J. Biol. Chem., Vol. 280, Issue 19, 18931-18942, May 13, 2005

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Palmitoylation and Intracellular Domain Interactions Both Contribute to Raft Targeting of Linker for Activation of T Cells^*

Hidehiko Shogomori, Adam T. Hammond¶, Anne G. Ostermeyer-Fay, Daniel J. Barr, Gerald W. Feigenson||, Erwin London, and Deborah A. Brown**

From the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, the ^¶Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, and the ^||Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

Received for publication, January 7, 2005 , and in revised form, March 4, 2005.

^* This work was supported by National Institutes of Health Grants GM47897 (to D. A. B.) and GM48596 (to E. L.), National Science Foundation Grant MCB-0315300 (to G. W. F.), and a grant from the W. M. Keck Foundation (to A. T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Sphingolipid Functions Laboratory, Supra-Biomolecular System Research Group, RIKEN Frontier Research System 2-1, Hirosawa, Wako Saitama 351-0198, Japan.

^** To whom correspondence should be addressed: Dept. of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215. Tel.: 631-632-8563; Fax: 631-632-8575; E-mail: deborah.brown{at}sunysb.edu.

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