K-Ras is a small G protein which localizes at the inner leaflet of the plasma membrane. The minimal membrane targeting signal of this protein consists of a polybasic carboxy-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 cellular membranes we first evaluated in vitro the effect of different electrolytes on membrane binding properties of recombinant K-Ras. Results from these experiments demonstrated the electrical and reversible nature of the K-Ras binding to biomembranes. To extent our analysis in a cellular system, we 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. Enzymatic remotion of sialic acid from the outer plasma membrane caused a redistribution of K-Ras from plasma membrane to a pericentriolar endosomal compartment. Inhibition of phosphatidylserine flipping out and synthesis of polyphosphoinositides at the inner plasma membrane by depleting cellular ATP resulted in a similar subcellular redistribution of the expressed K-Ras. Cell treatment with ionophores that modify the transmembrane potential also caused a rapid redistribution of K-Ras from plasma membrane to cytoplasm and endomembranes. Interestingly, Ca2+ ionophores caused a much more broad redistribution of K-Ras to endomembranes than K+ ionophores. Together, these results reveal the dynamic nature of the interactions between K-Ras and cellular membranes, and support the finding that cellular distribution of K-Ras is driven by electrostatic interactions of the polybasic domain of the protein with the negatively charged membranes.
