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. 2005 Feb;16(2):717–730. doi: 10.1091/mbc.E04-08-0740

Figure 2.

Figure 2.

Sla2p binding to liposomes in a PtdIns(4,5)P2 concentration-dependent and specific manner in vitro. (A) Sla2p binding to liposomes of various lipid compositions was tested. GST-Sla2p, 0.5 μM, was incubated with liposomes in a 70-μl reaction. Liposome-associated proteins were resolved by SDS-PAGE and were stained with SYPRO Red. PC/PE: 77 mol% DOPC, 22 mol% DOPE. Major-minor mix without PtdIns(4,5)P2: 51 mol% DOPC, 21 mol% DOPE, 9.8 mol% PI, 8 mol% DOPS, 5 mol% DOPA, 4.2 mol% PI4P, 2 mol% CDP-DAG. Major-minor mix with PtdIns(4,5)P2: 51 mol% DOPC, 21 mol% DOPE, 7.8 mol% PI, 8 mol% DOPS, 5 mol% DOPA, 4.2 mol% PI4P, 2 mol% CDP-DAG, 2 mol% PtdIns(4,5)P2. (B) Sla2p binding to liposomes composed of various PtdIns(4,5)P2 concentrations. Major-minor mix with (0.8-4 mol% PtdIns(4,5)P2): 51 mol% DOPC, 21 mol% DOPE, 9-5.8 mol% PI, 8 mol% DOPS, 5 mol% DOPA, 2.2 mol% PI4P, 2 mol% CDP-DAG, 0.8-4 mol% PtdIns(4,5)P. The Sla2p concentration in each assay was 0.5 μM. (C) Preferential binding of Sla2p to PtdIns(4,5)P2 versus PtdIns(3,5)P2. Liposomes made from Major-minor mix with 2 mol% PtdIns(4,5)P2 or 2 mol% PtdIns(3,5)P2 were tested for the binding of GST-Sla2p. The Sla2p concentration of each assay was 0.5 μM. (D) Binding of Sla2p to PtdIns3P, PtdIns4P, and PtdIns5P. Liposomes made from Major-minor mix with 2 mol% PtdIns(4,5)P2 or 2 mol% PtdIns3P, or PtdIns4P, or PtdIns5P. The Sla2p concentration in each assay was 0.5 μM.