Abstract:In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tappingmode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, whichsdepending on the concentration of the constituentssseparates into liquid-disordered (ld), liquid-ordered (lo), and solid-ordered (so) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquiddisordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is ld > lo . so. Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the ld/lo phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.

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