Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells.
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.
Shogomori
Hidehiko
H
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, USA.
Hammond
Adam T
AT
Ostermeyer-Fay
Anne G
AG
Barr
Daniel J
DJ
Feigenson
Gerald W
GW
London
Erwin
E
Brown
Deborah A
DA
D000818:Animals; D019556:COS Cells; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D008055:Lipids / Q000737:chemistry; D008213:Lymphocyte Activation; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D019308:Palmitic Acid / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012235:Rhodamines / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000378:metabolism*; D013696:Temperature
D003902:Detergents; D005456:Fluorescent Dyes; D008055:Lipids; D010455:Peptides; D012235:Rhodamines; D019308:Palmitic Acid; D017830:Octoxynol