GUVs were produced by a modification of the electroformation method24. The ‘Major-Minor mix' of lipids8 was prepared, consisting of the phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, 51mol%), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 22 mol%), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], 8 mol%), DOPA (1,2-dioleoyl-sn-glycero-3-phosphate, 5 mol%), soy PI (L-α-phosphatidylinositol, 8 mol%), porcine brain PI4P (L-α-phosphatidylinositol-4-phosphate, 2.2 mol%), porcine brain PI(4,5)P2 (L-α-phosphatidylinositol-4,5-bisphosphate, 0.8 mol%), CDP-DAG (1,2-dioleoyl-sn-glycero-3-(cytidine diphosphate), 2 mol%; all lipids from Avanti Polar Lipids, Alabaster, AL), fluorescent lipid analog (1 mol%, see below) and ergosterol (Sigma, St. Louis, MO; 20% ergosterol by weight, 80% phospholipids by weight). As fluorescent lipid analogs, DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, Invitrogen, Carlsbad, CA; for single-color confocal microscopy), DiDC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate, Invitrogen; for two-color confocal microscopy) and Texas-Red DHPE (Texas Red-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, Invitrogen) were used.