Structure and function of the C-terminal hypervariable region of K-Ras4B in plasma membrane targetting and transformation

The C-terminal hypervariable domain of K-Ras4B targets the protein to the p lasma membrane by a combination of positive charge and a hydrophobic signal (farnesyl group), We analysed the contribution of several structural featu res of the domain: net charge, charge distribution, amino acid sequence and lipid specificity to membrane targetting and function by using artificial 'hypervariable' domains fused to either EGFP or V12K-Ras4B, We found that c harge and a lipid residue are sufficient for plasma membrane localization a nd function of the constitutively active V12K-Ras4B. However, the amount of net charge, charge distribution and the length of the anchoring domain are important, Increasing the net charge and concentrating it close to the C-t erminus increases not only the percentage of membrane bound protein, but al so shifts the distribution from internal membranes, including the nuclear e nvelope, to the plasma membrane, While plasma membrane binding is necessary for V12K-Ras4B activity (MAPK activation and focus formation), we found th at there are additional restrictions. In particular, mutants with very high ly charged domains that bind almost exclusively to the plasma membrane show less transforming potential than expected. In addition, a construct with a short 'hypervariable' domain (7 amino acids) also has decreased transforma tion activity. These results suggest that specific interactions between K-R as4B and the plasma membrane are required.