We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADHI. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.We have been studying the two RAS genes, RASI and RAS2, of Saccsharony(ces cereivisiae as models for the mammalian ras oncogenes. The yeast RAS proteins are structurally, functionally, and biochemically similar to their mammalian counterparts and at least one of their effector systems is known (3,7,8,18,24,34,36,38). The yeast RAS genes were originally isolated by using mammalian ras genes to screen libraries from S. cerev,isiae (7,24). They encode proteins that are highly homologous to the mammalian ras proteins, particularly in their amino-terminal half. Both yeast and mammalian RAS proteins undergo similar processing events and localize to membrane fractions (6,11,25,30,42). All RAS proteins bind guanine nucleotides and possess an intrinsic GTPase activity. The GTPase activity is reduced in a number of oncogenic forms of mammalian ras proteins containing point mutations (12,21,29,33,34

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.

R190r0u5 feed6ack c0ntr01 0f cAMP 1eve15 1n 5acchar0myce5 cerev151aeJun-1ch1 N1kawa, 1 5c0tt Camer0n, 7aka5h1 70da, 2 Kenneth M. Fer9u50n, and M1chae1 W191erWe have 1501ated and character12ed n0rma1 and mutant a11e1e5 0f many 0f the 9ene5 0f the RA5/adeny1y1 cyc1a5e pathway 0f the yea5t 5acchar0myce5 cerev151ae. Man1pu1at10n 0f th05e 9ene5 ha5 revea1ed a 5y5tem f0r feed6ack c0ntr01 that can m0du1ate cAMP 1eve15 0ver at 1ea5t a 10,000-f01d ran9e. 7he feed6ack c0ntr01 depend5 up0n the act1v1ty 0f the cAMP-dependent pr0te1n k1na5e5 and re4u1re5 the pre5ence 0f the CDC25 and//A5 pr0te1n5.7he capac1ty f0r 5uch dramat1c c0ntr01 0f cAMP 1eve15 ra15e5 fundamenta1 4ue5t10n5 a60ut the n0rma1 mechan15m 0f act10n 0f the cAMP 519na11n9 5y5tem 1n yea5t.[Key W0rd5:5acchar0myce5 cerev151ae; cAMP; RA5/adeny1y1 cyc1a5e pathway; CDC25] F1uctuat10n5 1n cAMP c0ncentrat10n5 p1ay a maj0r r01e 1n m0du1at1n9 the affa1r5 0f eukary0t1c ce115.1n the yea5t 5acchar0myce5 cerev151ae, adeny1y1 cyc1a5e act1v1ty 15 dependent up0n the pre5ence 0f e1ther RA51 0r RA52 pr0te1n5, and the5e pr0te1n5 pre5uma61y m0du1ate adeny1y1 cyc1a5e (8r0ek et a1. 1985; 70da et a1. 1985). 70 1nve5t19ate the 1nteract10n 6etween adeny1y1 cyc1a5e and RA5 pr0te1n5, we and 0ther5 have c10ned and mutated many 0f the 9ene5 0f the RA5/adeny1y1 cyc1a5e pathway, 1nc1ud1n9 CDC25, the pr0duct 0f wh1ch pr06a61y m0du-1ate5 adeny1y1 cyc1a5e thr0u9h 1t5 effect5 0n RA5 pr0-te1n5 (8r0ek et a1. 1987; R061n50n et a1. 1987); RA51 and RA52, the tw0 h0m01095 0f the mamma11an ra5 0nc0-9ene5 (DeFe0-J0ne5 et a1. 1983; P0wer5 et a1. 1984); CYR1, the 9ene enc0d1n9 adeny1y1 cyc1a5e (Mat5um0t0 et a1. 1982(Mat5um0t0 et a1. , 1984 Kata0ka et a1. 1985); 7PK1, 7PK2, and 7PK3, three 9ene5 that enc0de the cata1yt1c 5u6un1t5 0f the cAMP-dependent pr0te1n k1na5e (cAPK) (70da et a1. 1987a); 8CY1, the 9ene enc0d1n9 the re9u1at0ry 5u6un1t 0f cAPK {70da et a1. 19876); and PDE1 and PDE2, the 9ene5 enc0d1n9 the 10w-and h19h-aff1n1ty cAMP ph05-ph0d1e5tera5e5, re5pect1ve1y (L0nde560r0u9h and 5u0r-anta 1983; 5u0ranta and L0nde560r0u9h 1984; 5a55 et a1. 1986; N1kawa et a1. 1987). Mea5urement5 0f cAMP 1eve15 1n ce115 w1th d15rupt10n5 0f the PDE 9ene5 1ed u5 t0 5u5pect the ex15tence 0f a ne9at1ve feed6ack mechan15m f0r c0ntr0111n9 cAMP 1eve15, 6ecau5e 1eve15 0f cAMP were 0n1y tw0-t0 three-f01d h19her than w11d type 1n 5tra1n5 1ack1n9 60th ph05ph0d1e5tera5e 9ene5 (N1kawa et a1. 1987). 7he exper1ment5 pre5ented here dem0n-5trate the ex15tence 0f 5uch a mechan15m. 5tud1e5 w1th mutant 7PK and 8CY1 9ene5 1nd1cate that the de9ree 0f feed6ack depend5 0n the act1v1ty 0f cAPK. 7he m0du1a-t10n 0f cAMP 1eve15 ach1eva61e 6y th15 mechan15m 5pan5 at 1ea5t f0ur 0rder5 0f ma9n1tude and 15 ent1re1y dependent up0n RA5 and CDC25 pr0te1n5. Re5u1t5Man1pu1at10n 0f cAMP 1eve15 1n ph05ph0d1e5tera5e-def1c1ent 5tra1n5.We have mea5ured cAMP 1eve15 1n yea5t 5tra1n5 def1-c1ent 1n cAMP ph05ph0d1e5tera5e {5ee 7a61e 1 f0r 5tra1n de5cr1pt10n5). Leve15 were mea5ured 6y rad101mmun0-a55ay 1n cu1ture5 9r0wn t0 fate exp0nent1a1 pha5e 1n r1ch med1u...

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

A novel gene, IRE1, of Saccharomyces cerevisiae was cloned through genetic complementation of a myoinositol auxotrophic mutant. The predicted amino acid sequence indicated that IRE1 encodes a protein of 126983 Da with two highly hydrophobic regions, probably a signal sequence and a membrane-spanning region. The carboxy-terminal region of IRE1 showed close sequence similarity to the catalytic domains of protein kinases. Disruption of the IRE1 locus caused myo-inositol auxotrophy. The IRE1 product is very likely a protein kinase required for myo-inositol synthesis.