Spatial localization of p21(ras) at the plasma membrane

Abstract

Post-translational modifications (farnesylation, proteolysis, and methylation) of the CAAX motif at the C-terminus and a second upstream signal (polybasic domain or palmitoylated cysteine residue) are believed to result in random association of p21ras with the plasma membrane. Replacement of the farnesyl group with a geranylgeranyl group results in association of p21ras with the plasma membrane, but geranylgeranylated p21v-ras is not transforming, suggesting that a specific recognition of p21ras by the plasma membrane is necessary for p21ras function. To examine structural aspects of the interaction between p21ras and the plasma membrane, immunofluorescence microscopy and electron microscopy experiments were performed using fibroblasts that over-express human p21H-ras. Immunofluorescent labeling of p21ras revealed an organized pattern of localization, suggesting that the interaction is not the result of simple insertion of hydrophobic side chains into the lipid bilayer. Furthermore, fluorescence recovery after photobleaching (FRAP) experiments indicate that p21H-ras is immobile at the plasma membrane. Immobility of p21H-ras in the plasma membrane is inconsistent with a model of random hydrophobic insertion into the lipid bilayer, again indicating that the association between p21H-ras and the plasma membrane involves molecular interactions beyond simple hydrophobic associations. Immunogold labeling of p21H-ras in electron micrographs revealed gold particles abundantly labeling protruding structures that form clusters at the plasma membrane. Within any cluster the protrusions are at least twice the diameter (>4 times the area) of the 10-nm gold particles, suggesting that the protrusions may be large complexes of proteins. Immunogold labeling of p21ras was never seen in areas of membrane without protrusions, which suggests that the protrusions are necessary for p21H-ras association with the plasma membrane. These results show that the lipid bilayer alone is not sufficient for p21H-ras association with the plasma membrane. When the plasma membranes were washed with low- and high-ionic strength buffers to remove peripheral membrane proteins, 95% of the fluorescent anti-p21ras labeling disappeared from the fluorescence images, and almost all of the immunogold p21ras labeling was eliminated in electron micrographs. This evidence supports the necessity for involvement of plasma membrane-associated proteins in p21ras localization to the plasma membrane.