Adenylyl Cyclase in Yeast

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYRl polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have determined hydrodynamic properties of yeast adenylyl cyclase in taurocholate extracts of wild type and RASdeficient membranes. In taurocholate extracts of both kinds of membranes, the enzyme is insensitive to guanine nucleotide stimulation; in the presence of 0.5 M NaCl, the taurocholate-solubilized enzyme has a sedimentation coefficient of 12.5 S and a Stokes radius of 11 nm, consistent with a molecular weight of 594,000 for the protein-detergent complex. Treatment of particulate fractions with trypsin (<lo pg/ml) markedly activates membrane-bound adenylyl cyclase activity, abolishes stimulation by guanine nucleotides, and reduces the sedimentation coefficient of the detergentsolubilized enzyme; higher concentrations of trypsin release a still smaller water-soluble enzyme complex (7.5 S, 6.1 nm Stokes radius, calculated M. = 190,000) from the membrane. In combination with genetic evidence (Kataoka, T., Broek, D., and Wigler M., (1985) Cell 43,493-505), our data are consistent with a structural and functional model of yeast adenylyl cyclase in which GTP-activated RAS proteins stimulate cAMP synthesis by relieving an inhibitory constraint on the activity of the CYRl gene product. This constraint may be mediated by the amino-terminal portion of the CYRl polypeptide.