Intermembrane cholesterol transfer: Role of sterol carrier proteins and phosphatidylserine

Abstract

The effect of phosphatidylserine and sterol carrier proteins on cholesterol exchange was determined using an assay not requiring separation of donor and acceptor membrane vesicles. Sterol carrier protein-2 (SCP2, also called nonspecific lipid transfer protein), but not fatty acid binding protein (FABP, also called sterol carrier protein), enhanced the initial rate of sterol exchange between neutral zwitterionic phosphatidylcholine small unilamellar vesicles (SUV) 2.3-fold. Phosphatidylserine at 10 mol% increased the initial rate of spontaneous and of SCP2-mediated (but not FABP-mediated) sterol exchange by 22% and 44-fold, respectively. The SCP2 potentiation of sterol transfer was dependent on SCP2 concentration and on phosphatidylserine concentration. The SCP2-mediated sterol transfer was inhibited by a variety of cations including KCl, divalent metal ions, and neomycin. The data suggest that SCP2 increase in activity for sterol transfer may be partly ascribed to charge on the phospholipid.

Shape and lipid-binding site of the nonspecific lipid-transfer protein (sterol carrier protein 2): A steady-state and time-resolved fluorescence study

“…In agreement with previous studies on the binding of fluorescently labeled PC and cholesterol analogues (Nichols et al, , 1988Schroeder et al, 1990), we have shown that nsL-TP is able to form a complex with PC. However in contrast to what was observed with PC-TP and PI-TP, this complex could not be isolated (data not shown).…”

“…This makes it very suitable for intrinsic t r y p tophan fluorescence measurements. Upon excitation (Aex = 280-300 nm), a broad emission spectrum is observed ranging from 300 to over 400 nm, with a maximum around 330-340 nm [see also Schroeder et al (1990)l. To assess the localization of the tryptophan residue, the tryptophan fluorescence of nsL-TP was quenched by addition of acrylamide or iodide.…”

“…This explains the impossibility of isolating the lipid/protein complex Crain & Zilversmit, 1980) and the inability of the protein to catalyze exchange between two separated monolayers (Van Amerongen et al, 1989). The apparent paradox between the low-affinity binding site of the protein (Nichols et al, 1988;Schroeder et al, 1990) on the one hand, and the complete immobilization of the DPH moiety upon binding of DPHp-PC and the inability to quench its fluorescence by water-soluble quenchers on the other hand, is explained by the great difference in time scales of the binding process (millisecond range) and the fluorescence process (nanosecond range). In fact, the immobilization of the DPH moiety on the DPH-fluorescence timescale indicates that the lifetime of the lipid/nsL-TP complex is longer than hundreds of nanoseconds.…”

“…Because nsL-TP contains a single tryptophan residue (Westerman & Wirtz, 1985) and is able to bind fluorescent lipid molecules (Nichols, , 1988Schroeder et al, 1990), it is very suitable for investigation by time-resolved and steady-state fluorescence spectroscopy. Time-resolved fluorescence spectroscopy is very powerful as it allows one to monitor the rotational dynamics of a protein molecule (Wolber et al, 1981;Visser et al, 1989).…”

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…The lifetime studies resolved for the first time at least two DHE domains in model membranes, one less sensitive to the aqueous than the other, and that these domains were dependent on the lipid composition, temperature, and other properties of the membrane (54,119). This possibility was further supported by model membrane studies of DHE exchange which provided kinetic evidence for the existence of multiple sterol containing domains in membranes (118,(121)(122)(123)(124). DHE was especially useful for these studies because the spontaneous exchange of DHE between model membranes was very similar to that of radiolabeled cholesterol (117).…”