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.

Differential solubilization of membrane components by cold 1% Triton X-100 extraction is common practice in cell biology and membrane research, used to define components of, or localization within membrane domains called lipid rafts. In this study, extraction of biological membranes was continuously monitored in single cells by confocal microscopy. The distributions of fluorescently-tagged proteins that label raft and non-raft membranes, cytosolic and cytoskeletal proteins were continuously monitored upon addition of the detergent. Membranes containing the non-raft membrane protein VSVG-GFP were immediately extracted from the plasma membrane, whereas raft-membrane proteins were predominantly resistant to the detergent. The morphological characteristics of differential membrane solubilization consisted of the formation of pores that expand and percolate as the detergent-mediated solubilization proceeds. Pore expansion and percolation was much slower and more restricted in non-polarized MDCK cells than in COS-7 cells. Heterologous overexpression in COS-7 cells of the fluorescently-tagged human MAL, a tetra-spanning, lipid-raft-associated protein, significantly slowed and limited membrane pore expansion and percolation. Extensive percolation resulting in large holes in the membrane was observed for the raft-associated, GPI-GFP-labeled membranes in COS-7 cells. Quantitative analysis carried out using pixel intensity variance as an indicator of membrane pore expansion demonstrated that the MAL protein is capable of modifying the plasma membrane, thereby increasing its resistance to detergent-induced pore formation.

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