Charged membrane surfaces impede the protein-mediated transfer of glycosphingolipids between phospholipid bilayers

A lipid transfer protein that facilitates the transfer of glycolipids betwe en donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa , pI 9.0) catalyzes the high specificity transfer of lipids that have sugar s beta-linked to either a ceramide or a diacylglycerol backbone, such as si mple glycolipids and gangliosides, but not the transfer of phospholipids, c holesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled g alactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of ne gatively charged lipid at 5 or 10 mol % into the donor vesicles significant ly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was no t as effective at slowing the transfer rate as was negative charge in the d onor vesicle. Increasing the ionic strength of the buffer with NaCl signifi cantly reversed the charge effects. At neutral pH, the transfer protein (pI congruent to 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-orde r kinetics and that the off-rate of the transfer protein from the donor ves icle surface is the rate-limiting step in the transfer process.