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Palmitoylation-Dependent Plasma Membrane Transport but Lipid Raft-Independent Signaling by Linker for Activation of T Cells¹

Matthias Hundt^*, Yohsuke Harada^*, Lauren De Giorgio^*, Natsuko Tanimura, Weiguo Zhang and Amnon Altman^2,*

^* Division of Cell Biology, La Jolla Institute for Allergy & Immunology, La Jolla, CA 92037; Division of Infectious Genetics, The Institute of Medical Science, University of Tokyo, Tokyo, Japan; and Department of Immunology, Duke University Medical Center, Durham, NC 27710

Linker for activation of T cells (LAT) is a dually palmitoylated transmembrane adaptor protein essential for T cell development and activation. However, whether LAT palmitoylation and/or lipid raft localization are required for its function is controversial. To address this question, we used a combination of biochemical, imaging, and genetic approaches, including LAT retrovirus-transduced mouse T cells and bone marrow chimeric mice. A nonpalmitoylated, non-lipid raft-residing mutant of transmembrane LAT could not reconstitute T cell development in bone marrow chimeric mice. This mutant was absent from the plasma membrane (PM) and was restricted mainly to the Golgi apparatus. A chimeric, nonpalmitoylated LAT protein consisting of the PM-targeting N-terminal sequence of Src kinase and the LAT cytoplasmic domain (Src-LAT) localized as a peripheral membrane protein in the PM, but outside lipid rafts. Nevertheless, Src-LAT restored T cell development and activation. Lastly, monopalmitoylation of LAT on Cys²⁶ (but not Cys²⁹) was required and sufficient for its PM transport and function. Thus, the function of LAT in T cells requires its PM, but not raft, localization, even when expressed as a peripheral membrane protein. Furthermore, LAT palmitoylation functions primarily as a sorting signal required for its PM transport.

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.

¹ This work was supported by National Institutes of Health Grant AI081078 and by institutional funds (to A.A.), and by a Leukemia & Lymphoma Society Special Fellowship (to M.H.).

² Address correspondence and reprint requests to Dr. Amnon Altman, Division of Cell Biology, La Jolla Institute for Allergy & Immunology, 9420 Athena Circle, La Jolla, CA 92037. E-mail address: amnon{at}liai.org

³ Abbreviations used in this paper: BM, bone marrow; B6 (mice), C57BL/6 mice; DAPI, 4',6'-diamidino-2-phenylindole; DN, double negative; DRM, detergent-resistant membrane; ICS, intracellular staining; IS, immunological synapse; LAT, linker for activation of T cells; LAX, linker for activation of X cells; PAT, protein acyl transferase; PM, plasma membrane; SEE, Staphylococcus enterotoxin E; TM, transmembrane; TRAP, transmembrane adaptor protein; wt, wild type; Int, intensity.