Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal...

We have studied the post-translational processing of p21ras proteins. The primary translation product pro-p21 is cytosolic and is rapidly converted to a cytosolic form (c-p21) of higher mobility on SDS-PAGE. c-p21 is converted in turn to the membrane-bound mature palmitoylated form (m-p21) of slightly higher mobility. These processing steps are accompanied by increases in isoelectric point and in hydrophobicity as judged by Triton X-114 partitioning. Although the increases in electrophoretic mobility and hydrophobicity precede acylation we show that mutation of Cys186, which has been shown to block acylation, also abolishes the pro-p21 to c-p21 conversion. Thus the Cys186 residue is involved in the processing steps prior to acylation. We have identified two processing events which contribute to the pro-p21 conversion. Site-directed mutagenesis to insert tryptophan, which is not present in the wild type, followed by metabolic labelling with [3H]tryptophan has allowed us to map a proteolytic processing event which removes the three C-terminal residues. In addition, both the c-p21 and m-p21 forms are carboxyl-methylated. Approximately one methyl group is incorporated per molecule of p21 at steady state, which can partially account for the increase in isoelectric point. Unlike palmitate, methyl group turnover is not observed.