K-Ras is a small G protein which mainly localizes at the inner leaflet of the plasma membrane regulating cellular growth, differentiation and apoptosis. The membrane targeting signal of this protein consists of a polybasic C-terminal sequence of six contiguous lysines and a farnesylated cysteine. Results from biophysical studies in model systems suggest that hydrophobic and electrostatic interactions are responsible for membrane binding properties of K-Ras. To test this hypothesis in a cellular system, we evaluated in vitro the effect of electrolytes on membrane binding properties of K-Ras. Results demonstrated the electrical and reversible nature of the K-Ras binding to anionic lipids in biomembranes. We next investigated membrane binding and subcellular distribution of K-Ras after perturbing the electrical properties of the outer and inner leaflet of plasma membrane and the ionic gradients through it. Remotion of sialic acid and inhibition of polyphosphoinositides synthesis at the plasma membrane caused an accumulation of K-Ras to recycling endosome membranes. Cell treatment with Ca2+ and K+ ionophores that modify the transmembrane potential caused a redistribution of K-Ras to cytoplasm and endomembranes. Together, these results reveal the dynamic nature of the interactions between K-Ras and cellular membranes, and support the finding that subcellular distribution of K-Ras is driven by electrostatic interactions of the polybasic domain of the protein with the negatively charged membranes.
