PDEl and PDEZ

The Saccharomyces cereuisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added CAMP. Upon the addition of CAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDEl and PDEZ, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYRI , which encodes adenylate cyclase, and the slow growth phenotype caused by the cyrl mutation was suppressed by the pde2 mutation. Therefore P28-24C is fortuitously a pdel pde2 cyrl triple mutant. Responsiveness to cAMP conferred by p d e mutations suggests that S. cereuvisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases. G ENETIC study of the cAMP pathway in the yeast Saccharomyces cerevisiae was initiated by the isolation of CAMP-requiring mutants, which depend on exogenously added cAMP for growth (MATSUMOTO et al. 1982b). These mutants were isolated from a strain carrying the caml, cam2 and cam3 mutations, which enable cells to utilize exogenous cAMP as an adenine source and were considered to facilitate cAMP uptake (MATSUMOTO et al. 1982a). The cam mutations, however, were found not to be necessary for CAMPdependent growth of cyrl-1 mutants with the P-2824C genetic background (MATSUMOTO et al. 1982b). Here I report that the responsiveness to exogenously added cAMP of strain P-28-24C results from mutations in the cAMP phosphodiesterase genes. MATERIALS AND METHODS Strains: Yeast strains used in this study are listed in Table 1. A cyrI-230 strain, HM110-3B, was constructed by five serial crosses of HM57-2C to YPH252. Gene disruptions of PDEl and PDE2 were carried out by one-step gene replacement (ROTHSTEIN 1983) using plasmids ppde1::URAS and ppdeQ::HIS3, respectively (NIKAWA et al. 1987). Strains carrying a deletion allele of CYRl were constructed by transplacement (WINSTON, CHUMLEY and FINK 1983). A URA3 plasmid carrying a CYRl gene with an internal deletion (MORISHITA, MATSUURA and UNO 1993) was integrated at the CYRl locus of ura3 strains after digestion with ClaI, yielding strains carrying URA3 flanked by intact and deletion alleles of CYRl . 5-Fluoroorotic acid resistant derivatives from these strains were screened for CAMP-dependent growth, yielding strains carrying only the deletion allele of CYR I. University of California, Los Angeles, California 90024. Genetics 135: 321-326 (October, 1993) ' Present address: Department of Microbiology and Molecular Genetics, Media: Complete medium (YPD), synthetic minimal medium (SD) supplemented with nutrients, and minimal sporulation medium were prepared as described (SHERMAN, FINK and HICKS 1986; SHERMAN 1991). Synthetic complete medium lacking uracil was prepared by supplementing SD with tryptophan, adenine, and 0.5% casamino acids. YPD medium containing 1 or 5 mM cAMP was prepared by adding a filter-sterilized 50 mM cAMP solution, the pH of which was adjusted to 5.6 with NaOH. Medium containing 5-fluoroorotic acid was prepared as described (BOEKE et al. 1987). Cells were grown at 30" unless noted otherwise. General genetic methods and transformation: Standard methods were used for genetic analysis (SHERMAN, FINK and HICKS 1986; SHERMAN 1991). Yeast transformation was carried out by the lithium acetate method (ITO et al. 1983). Assay of responsiveness to CAMP: Since mutants with elevated activity of the cAMP pathway lose viability rapidly, colonies or patches of such mutants show a red stain on a YPD plate containing 20 pg/ml phloxine B, which stains dead cells (CANNON, GIBBS and TATCHELL 1986). The ability of cells to respond to exogenously added cAMP was tested by phloxine B staining. A red stain with the dye upon the addition of cAMP indicates responsiveness to CAMP. Incubation at 30" for 2 days on YPD medium containing phloxine B did not allow the development of red color by ade2. Assay of glycogen accumulation: Relative glycogen levels were measured by iodine staining with a solution of 0.2% I p , 0.4% KI (CHESTER 1968).