Sterol Carrier Protein-2 Selectively Alters Lipid Composition and Cholesterol Dynamics of Caveolae/Lipid Raft vs Nonraft Domains in L-Cell Fibroblast Plasma Membranes

Abstract

Although the functional significance of caveolae/lipid rafts in cellular signaling and cholesterol transfer is increasingly recognized, almost nothing is known regarding the lipids, cholesterol dynamics, and factors regulating these properties in caveolae/lipid rafts as opposed to nonlipid raft domains of the plasma membrane. The present findings demonstrate the utility of con-A affinity chromatography for simultaneous isolation of caveolae/lipid raft and nonlipid raft domains from plasma membranes of L-cell fibroblasts. These domains differed markedly in both protein and lipid constituents. Although caveolae/lipid rafts were enriched in total lipid, cholesterol, and phospholipid as well as other markers for these domains, the cholesterol/phospholipid ratio of caveolae/lipid rafts did not differ from that of nonlipid rafts. Nevertheless, spontaneous sterol transfer was 7-12-fold faster from caveolae/lipid raft than nonlipid raft domains of the plasma membrane. This was largely due to the near absence of exchangeable sterol in the nonlipid rafts. SCP-2 dramatically and selectively enhanced sterol transfer from caveolae/lipid rafts, but not from nonlipid rafts. Finally, overexpression of SCP-2 significantly altered the sterol dynamics of caveolae/lipid rafts to facilitate retention of cholesterol within the cell. These results established for the first time that (i) caveolae/lipid rafts, rather than the nonlipid raft domains, contain significant levels of rapidly transferable sterol, consistent with their role in spontaneous sterol transfer from and through the plasma membrane, and (ii) SCP-2 selectively regulates how caveolae/lipid rafts, but not nonlipid raft domains, mediate cholesterol trafficking through the plasma membrane.

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…Thus, DHE with its low quantum yield can easily be detected at the plasma membrane ( Fig. 6B: inset) and in intracellular compartments as had been observed from previous spectroscopic studies (8,70,102,135,172). Signal saturation may occur as a result of accumulation of unesterified or esterified monomeric DHE in lipid storage vesicles (Fig.…”

“…The naturally-occurring fluorescent sterol DHE (19,21) has proven a powerful probe for use in investigating membrane structure and cholesterol domain dynamics in vitro (8,54,55,65,70,102,172), for examining cholesterol-protein interactions in vitro (2,39,96,101,102,108,205), and for first time visualization in real-time of the distribution of cholesterol in the plasma membranes in living cells (70,203,204). Successful application of the fluorescent DHE to investigation of the function and organization of sterols in membranes, especially lipid raft/ caveolae microdomains of living cells, requires the use of highly purified DHE.…”

“…The finding of rapid transbilayer sterol movement suggested that the asymmetric transbilayer distribution in the plasma membrane was not due to restricted movement of cholesterol from one leaflet to the other, but was instead a property of the lipid bilayer itself. DHE transbilayer distribution has broad applicability in studies of the action of anesthetics, cholesterol lowering drugs (statins), alcohol (chronic and acute), lupus erythematosus, aging, apoE, and sterol carrier proteins all of which can alter plasma membrane or synaptosomal plasma membrane transbilayer sterol distribution and/or transbilayer fluidity (26,135,136,145,149,150,154,(161)(162)(163)(164)(165)(166)(167)(168)(169)(170)(171)(172).Second, the DHE lateral distribution in both model and biological membranes was not uniform, but instead reflected sterol-rich and poor domains (rev. in (54,55).…”

“…micro-crystals, lipoproteins, LUV, or cyclodextrin complexes) may affect the distribution of fluorescent sterol to lipid rafts/caveolae or non-rafts (see Section 6.3 and respectively (26,27,70,136). DHE was codistributed similarly as cholesterol into sterol-rich and -poor domains (26,27,70,82,102,135,136,172,182,196). Lipid raft/caveolae associated receptors such as the oxytocin receptor are sensitive to the structure of the sterol with which it interacts and both cholesterol and DHE, but not other sterols, are effective in reconstituting activity of this highly sterol-sensitive plasma membrane protein in cholesterol-depleted lipid rafts/caveolae (89-91).…”

“…HDL) and endogenous (e.g. SCP-2) cholesterol binding proteins preferentially donate or extract cholesterol from cholesterol-rich, but not cholesterol-poor, microdomains (26,27,136,137,172). The microdomain/lipid raft concept, despite controversy in details, provides a framework for McIntosh et al …”