Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells

Abstract

We have analyzed the guanine nucleotides bound to mammalian ras and yeast RAS proteins overexpressed in [32P]orthophosphate-labeled cultures of exponentially growing Saccharomyces cerevisiae cells. Whereas S. cerevisiae RAS1 and RAS2 proteins were immunoprecipitated bound entirely to GDP, mammalian Harvey ras was isolated with GTP and GDP bound in near-equimolar proportions. In a strain overexpressing a RAS2 variant where the RAS unique C-terminal domain was deleted, both GTP and GDP were detected in a ratio of 3:97. Increased amounts of GTP (16-75% of total guanine nucleotide) were observed bound to all ras proteins containing mutations that inhibit GTP hydrolytic activity. Increasing proportions of GTP bound to the various ras proteins correlated with increasing biological potency to bypass cdc25 lethality in yeast.