title: Membrane raft association is a determinant of plasma membrane localization.
abstract: The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030;
authors.forename: Blanca B
authors.initials: BB
authors.lastname: Diaz-Rohrer
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030;
authors.forename: Kandice R
authors.initials: KR
authors.lastname: Levental
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany; and.
authors.forename: Kai
authors.initials: K
authors.lastname: Simons
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030;Cancer Prevention and Research Institute of Texas, Austin, TX 78701 ilya.levental@uth.tmc.edu.
authors.forename: Ilya
authors.initials: I
authors.lastname: Levental
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054878:Lipoylation; D008247:Lysosomes / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D022161:Transport Vesicles / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C491733:Lat protein, rat; D008565:Membrane Proteins; D010750:Phosphoproteins; D049452:Green Fluorescent Proteins
keywords: endocytosis; membrane domain; microdomains; phase separation; trafficking