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* Instituto de Investigaciones Bioquímicas, Consejo Nacional de Investigaciones Cientificas y Technológicas-Universidad Nacional de La Plata, La Plata, Argentina, 1900
Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, Urbana, IL 61801
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801
1 To whom correspondence should be addressed. e-mail: aletricerri{at}yahoo.com
Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down.
Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.
Abbreviations: Å, angstrom; apoA-I, apolipoprotein A-I; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; GP, general polarization; GUV, giant unilamellar vesicle; Laurdan, 6-dodecanoyl-2-dimethyl-amino-naphthalene; MLV, multilamellar vesicle; PAGGE, polyacrylamide gradient gel electrophoresis; PC, phosphatidylcholine; PL, phospholipid; POPC, 1-palmitoyl, 2-oleoylphosphatidylcholine; Pt, Platinum; SUV, small unilamellar vesicle; TSB, Tris salt buffer
Supplementary key words giant unilamellar vesicles • small unilamellar vesicles • lipid-protein interactions • lipid-phase coexistence
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