Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

Abstract

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.

Selective export of human GPI-anchored proteins from the endoplasmic reticulum

“…Alternatively, the interaction of GPI-anchored proteins and p24-p23 is mediated by a linker protein. Such a function has been proposed for VIP17/MAL in sorting GPI-anchored proteins to apical transport vesicles in polarized cells (Magal et al, 2009;Schuck and Simons, 2004). Perhaps PGAP5 is such a linker in the ER.…”

MAL Protein Controls Protein Sorting at the Supramolecular Activation Cluster of Human T Lymphocytes

“…Therefore, the distribution of highly ordered membranes at the cSMAC and that of the raft-associated proteins Lck and LAT are strictly dependent on MAL targeting to the cSMAC. The proposed role of MAL as machinery for membrane order organization at the IS is supported by the observation of that MAL clustering creates specifically large, condensed membrane domains in the plasma membrane (20).…”

“…Remarkably, the effect on HA and Lck targeting strictly correlates with the exclusion of both cargo molecules from DRMs. MAL clustering has been shown to create specifically large, condensed membrane domains in the plasma membrane (20). MAL is, therefore, considered a component of the machinery for organization and function of condensed membranes in epithelial cells and human T lymphocytes (17,20).…”

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation

“…Given that the possible effect of FAPP2 KD on the condensation of membranes at the base of the primary cilium was not addressed, we do not know whether FAPP2 is also important for proper membrane condensation at the ciliary base. Given the role of MAL in the transport of raft-associated molecules (Anton et al, 2011;Cheong et al, 1999;Puertollano et al, 1999), it is plausible that MAL affects membrane condensation at the ciliary base through the supply of raft membranes, although a direct role of MAL in the organization of raft lipids is also feasible (Magal et al, 2009). One interesting possibility is that the condensed membranes at the periciliary region are required for efficient assembly of machinery for primary cilium growth.…”