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Abstract
Ras GTPases are crucial for growth signaling in eukaryotes and are found mutated in 30% of human cancers. Ras isoforms undergo multiple post-translational modifications (PTMs) at their carboxyl-termini. These include 3 steps of CAAX (cysteine-aliphatic-aliphatic-one of several (X) residues) motif modifications (i. cysteine isoprenyl (farnesyl) lipidation, ii. endoproteolysis of ‘AAX’, and iii. carboxylmethylation of the lipidated cysteine), and palmitoyl lipidation near the CAAX motif in some Ras isoforms. While these PTMs together are known to aid Ras membrane association, the individual contributions of the PTMs to Ras localization and signaling are less clear. Specifically, the role of Ras CAAX proteolysis is not well understood due to the absence of an effective strategy to generate non-proteolyzed Ras without affecting other CAAX proteins in the cell. In this study, to understand the impact of loss of CAAX proteolysis on Ras function, we investigated the properties of a farnesylated and non-proteolyzed Ras variant (i.e., shunted Ras) bearing the CASQ motif. Importantly, this method maintains normal CAAX PTM in other proteins, which enables effects to be specifically attributed to Ras. In both Saccharomyces cerevisiae (i.e., budding yeast) and mammalian cells, we observed phenotypes associated with decreased signaling from shunted hyperactive Ras mutants (ScRas2-G19V and HsNRas-G12V, respectively). We also determined that one of either CAAX proteolysis or palmitoylation is sufficient for fully sustaining yeast viability by wildtype (non-hyperactive) farnesylated ScRas2, whereas the absence of both CAAX proteolysis and palmitoylation modifications decreased viability by approximately 70%, perhaps suggesting a shared role for the modifications. Further, in yeast, we found effects of ScRas2 CAAX proteolysis and palmitoylation on localization, protein levels, and GTP activation, suggesting that the impact of Ras carboxyl-terminal PTMs might extend beyond the previously well-appreciated regulation of membrane association. Finally, we propose that this strategy employed to shunt Ras could be easily applied to other CAAX proteins for examining the specific impact of CAAX proteolysis on their functions.