J. Colicelli

Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein

Adenylyl cyclase from S. cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which we now call CAP (cyclase-associated protein). We cloned a cDNA encoding CAP by screening a yeast cDNA expression library in E. coli with antisera raised against the purified protein. The cDNA contained an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases. Adenylyl cyclase activity in membranes from cells that lacked CAP was not stimulated by RAS2 proteins in vitro. These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system. Mutants lacking CAP had four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology.

Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes.

Studies of RAS Function in the Yeast Saccharomyces cerevisiae

The three mammalian RAS genes, Ha-ras, Ki-ras, and N-ras, are capable of the malignant transformation of cultured animal cells (Barbacid 1987). Mutations in these genes have been linked to a large number of human cancers (Barbacid 1987). These genes encode closely related proteins that bind guanine nucleotides (Scolnick et al. 1979; Shih et al. 1980; Ellis et al. 1981) and are localized to the inner surface of the plasma membrane (Willingham et al. 1980; Papageorge et al, 1982). Normal RAS proteins also slowly hydrolyze GTP (Gibbs et al. 1984; McGrath et al. 1984; Sweet et al. 1984). These properties are similar to those of the G proteins, which has led to the widespread expectation that RAS proteins, like G proteins, are involved in the transduction of membrane signals that are linked to cellular proliferation or differentiation.

Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase.

Large deletion and small insertion mutations in the adenylyl cyclase gene of Saccharomyces cerevisiae were used to map regions required for activation by RAS protein in vitro. The amino-terminal 605 amino acids were found to be dispensable for responsiveness to RAS protein. All other deletions in adenylyl cyclase destroyed its ability to respond to RAS. Small insertion mutations within the leucine-rich repeat region also prevented RAS responsiveness, while other insertions did not.