Sorting of tN-Ras by Membrane Curvature in Lipid Vesicles and Tubes

Abstract

Ras proteins are small GTPases that are post-translationally modified by the attachment of lipid moieties (1). This modification is essential for the correct trafficking and sorting of Ras proteins through the vesicular pathway from the Golgi to the plasma membrane (2).Traditionally the sorting of Ras is primarily discussed in the context of membrane domains in flat membranes, neglecting the influence of membrane shape. Recently we have demonstrated, utilizing our single liposome curvature (SLiC) assay that the minimal anchoring motif of N-Ras (tN-Ras) up-concentrates in areas of high membrane curvature (not published), suggesting that curvature might act as a cue for the spatial localization of Ras proteins.In the SLiC assay, curvature-sensing molecules are added from aqueous solution to vesicles of different curvatures, but the vesicles are not in diffusive contact (3, 4). In vivo Ras proteins are anchored to membranes and laterally sorts between curved and planar membranes, which are in diffusive contact. To study the curvature-sensing ability of tN-Ras in a setup mimicking the in vivo scenario we developed a membrane tube based assay in which the tubes are in diffusive contact with a lipid bilayer.Membrane tubes are formed by heating a confined lipid bilayer (5). The tubes eventually adsorb to the flat membrane, which enable imaging by confocal fluorescence microscopy. After addition of tN-Ras we observed a preferential sorting into curved tubes rather than the flat bilayer. This observation further implies a pivoting role of membrane shape as a regulator of Ras-protein localization.1. Prior & Hancock, Semin Cell Dev Biol 23:145(2012).2. Choy et al., Cell 98:69(1999).3. Kunding et al., Biophysical Journal 95:1176(2008).4. Hatzakis et al., Nat Chem Biol 5:835(2009).5. Weirich & Fygenson, Poster Abstract 2731, Biophys. Soc. 2011.