RESUMO
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.
Assuntos
Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Mielina/metabolismo , Proteolipídeos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Humanos , Lipídeos de Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas da Mielina/química , Proteínas da Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Conformação Proteica , Proteolipídeos/química , Proteolipídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The secretory membrane system is comprised of membrane-bound organelles defined by specific sets of proteins that function in sequential modification of cargo proteins and lipids. This processing of cargo proteins and lipids is coupled to their secretory transport. Here, we investigated the effect of inhibiting N-glycan processing by swainsonine, an inhibitor of Golgi alpha1,2-mannosidase-II, on secretory transport of the thermo-reversible tsO45 mutant of vesicular stomatitis virus glycoprotein tagged with green fluorescent protein (VSVG-FP). Quantitative analysis using kinetic modeling combined with live cell imaging was used to derive the rate coefficients that delineate secretory transport of VSVG-FP. We found that neither inhibition of N-glycan processing nor elimination by mutagenesis of the first of the two asparagine-linked glycans had any significant effect on the rate of VSVG-FP transport through the Golgi. These data suggest that at least for VSVG, the multi-enzymatic process of N-glycan modification does not comprise a rate-limiting step for its Golgi efflux.
