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Vol. 20, Issue 16, 3751-3762, August 15, 2009

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Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

Lee Goldstein Magal^*, Yakey Yaffe^*, Jeanne Shepshelovich^*, Juan Francisco Aranda, Maria del Carmen de Marco, Katharina Gaus, Miguel Angel Alonso, and Koret Hirschberg^*

*Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel; Centro de Biologia Molecular "Severo Ochoa," Universidad Autonoma de Madrid, Consejo Superior de Investigaciones Cientificas, Madrid 28049, Spain; and Centre for Vascular Research, University of New South Wales and the Department of Haematology, Prince of Wales Hospital, Sydney 2052, Australia

Submitted February 18, 2009; Revised June 10, 2009; Accepted June 11, 2009
Monitoring Editor: Jean E. Gruenberg

MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft–mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures—spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL—laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via xx intramembrane protein–protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms.

Address correspondence to: Koret Hirschberg (koty{at}post.tau.ac.il)

Abbreviations used: ER, endoplasmic reticulum; FP, fluorescent protein; GP, general polarization; GPI, glycophosphatidylinositol; OC, ordered cluster; PM, plasma membrane; TMD, transmembrane domain.

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