Lipid-Anchored Ras is Sorted by Membrane Curvature Both In Vitro and in Living Cells

Abstract

In vivo studies have reported preferential partitioning of Ras GTPases into ordered lipid-protein membrane domains, a process believed to regulate both cellular signaling and protein trafficking.1 However studies in vitro have failed to quantify a preferential partitioning of full length Ras proteins into the liquid ordered phase2,3 and thus a biophysically validated mechanism for in vivo sorting of Ras is still missing. We recently showed that lipidated proteins localize to highly curved membranes in vitro.4 Here we study both in vitro and in vivo whether recruitment by membrane curvature can sort full length lipid-anchored Ras.We employ a single vesicle fluorescence based assay to quantify in vitro the sorting by membrane curvature of full-length Ras proteins. We demonstrate a more than 50 fold increase in protein density on membranes of high curvature as compared to the density on flat membranes. To test for recruitment by membrane curvature in vivo we utilize hypo-osmotic swelling of cells, which flattens curved membrane regions. By measuring the local protein density using FRET,5 we detect a significant reduction in the clustering of Ras and other lipidated proteins upon membrane flattening. This demonstrates that recruitment by membrane curvature can sort Ras and potentially other lipidated proteins in cellular membranes. Furthermore sorting by membrane curvature constitutes the first biophysical sorting mechanism for Ras validated by both in vitro and in vivo measurements.1 Hancock, J. F. Nat. Rev. Mol. Cell Biol.4 (2003).2 Johnson, S. A. et al.Biochim. Biophys. Acta - Biomembranes1798 (2010).3 Nicolini, C. et al.J. Am. Chem. Soc.128 (2006).4 Hatzakis, N. S. et al.Nat. Chem. Biol.5 (2009).5 Kohnke, M. et al.Chem. Biol.19 (2012).