No abstract

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2vad9, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These results therefore imply that the effects of RAS2vi19 are mediated through its changes in cAMP concentration.Our laboratory group has been studying the mechanism of growth control in the yeast Saccharomyces cerevisiae with particular concentration on the functions of the RAS] and RAS2 genes, which are structurally and functionally homologous to the ras oncogenes of mammalian cells (1-4). At least one RASI or RAS2 gene is required for the continued growth of yeast cells (5, 6) and it has been shown that RAS genes are essential controlling elements for adenylate cyclase in yeast (2,7,8). A mutant RAS2 gene has been constructed that encodes valine at the 19th codon position instead of glycine (5). This mutant (RAS2vall9) is analogous to the mutant and oncogenic human Ha-ras gene, which was first recognized in the T24/EJ bladder cell line (9-11). Yeast cells that express the mutant RAS2vall9 gene fail to synthesize glycogen, show an abnormal sensitivity to starvation (8), show a defective ability to arrest in the G1 phase of the cell cycle (8), and are sensitive to heat shock (unpublished results). To better understand the mechanism of these effects, we have searched for yeast genes that, when present in high copy, reverse these phenotypic effects. One such gene has been found, and it encodes the high-affinity cAMP phosphodiesterase (PDEase) of S. cerevisiae. We here present the nucleotide sequence of this gene and describe some of the phenotypic consequences of its perturbation.

A new gene, SCH9, was isolated from Saccharomyces cerevisiae by its ability to complement a cdc25^^ mutation. Sequence analysis indicates that it encodes a 90,000-dalton protein with a carboxy-terminal domain homologous to yeast and mammalian cAMP-dependent protein kinase catalytic subunits. In addition to suppressing loss of CDC25 function, multicopy plasmids containing SCH9 suppress the growth defects of strains lacking the RAS genes, the CYRl gene, which encodes adenylyl cyclase, and the TPK genes, which encode the cAMP-dependent protein kinase catalytic subunits. Cells lacking SCH9 grow slowly and have a prolonged Gi phase of the cell cycle. This defect is suppressed by activation of the cAMP effector pathway. We propose that SCH9 encodes a protein kinase that is part of a growth control pathway which is at least partially redundant with the cAMP pathway.

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterases. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDEI gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDEI or PDE2 can reverse the growth arrest defects of yeast cells carrying the jRA2Val19 mutation. PDEI and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S.cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAu2Val-19 mutation. pdel-pde2-rasl-ras2-cells are viable.We have been investigating the pathways of growth regulation in the yeast Saccharomyces cerevisiae, particularly those pathways which involve the RAS proteins. The RAS] and RAS2 genes of S. cerevisiae are structurally and functionally closely related to the mammalian ras oncogenes (10,11,17,29). In S. cerevisiae, RAS proteins modulate adenylate cyclase in a GTP-dependent manner (4, 37). Yeast cells have severe defects in growth control when they lack RAS genes or contain RAS2 mutations analogous to those which activate the oncogenic properties of the mammalian RAS genes (18,35). In particular, yeast cells containing the