NOVEL DETERMINANTS OF H-RAS PLASMA-MEMBRANE LOCALIZATION AND TRANSFORMATION

Although it is well-established that modification of Ras by farnesol i s a critical step for its membrane association and transforming activi ty, the contribution of other C-terminal sequences and palmitate modif ication to Ras localization and function remains unclear, We have char acterized H-Ras mutant proteins with alterations in the palmitoylated cysteines or in sequences flanking these residues, We found that non-p almitoylated proteins were impaired not only in membrane association b ut also in transforming activity, Mutations which drastically altered residues adjacent to the palmitoylated cysteine did not abolish palmit oylation, However, despite continued lipid modification the mutant pro teins failed to bind to plasma membranes and instead accumulated on in ternal membranes and, importantly, were not transforming, Addition of an N-terminal myristoylation signal to these defective mutants, or to proteins entirely lacking the C-terminal 25 residues restored both pla sma membrane association and transforming activity, Thus, H-Ras does n ot absolutely require prenylation or palmitoylation nor indeed its hyp ervariable domain in order to interact with effecters that ultimately cause transformation, However, in this native state, the C-terminus ap pears to provide a combination of lipids and a previously unrecognized signal for specific plasma membrane targeting that are essential for the correct localization and biological function of H-Ras.