Figure 2.

Lipid requirements for AP-3 binding to chemically defined liposomes. Liposomes (200 μg/ml) prepared from various combinations of purified phospholipids as described below were assayed for the binding of AP-3 and COPI. Liposomes were incubated with 5 mg/ml gel-filtered bovine adrenal cytosol in the presence or absence of 100 μM GTPγS as described in Figure 1. Liposomes prepared from DOPC/DOPE/cholesterol (50:40:10, wt/wt) were designated as control liposomes (CONT). In liposomes containing additional phospholipids, the DOPE fraction was adjusted accordingly. Liposomes designated as PA, PS, and PI contained 10% of each phospholipid added to control liposomes. Liposomes denoted as PI4P and PIP2 contained 5% of each phospholipid added to control liposomes, and liposomes identified as PIP3 contained 2.5% PIP3 added to control liposomes. Liposomes prepared from soybean lipids containing 20% PC are denoted as SOY. Liposome-bound proteins were separated by SDS-PAGE, and binding of β-COP (detected with mAb M3A5) and ς3 was determined by immunoblotting and densitometry. Coat protein binding in the absence of GTPγS was considered to be nonspecific.