Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26

Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation.     

A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose- and acidification-induced cAMP signalling and not the basal cAMP level

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.     

Intracellular cAMP in Dictyostelium discoideum: Characterization of an aggregation-deficient mutant with elevated cAMP pools

Forty aggregation-deficient mutants of Dictyostelium discoideum were screened for changes in intracellular cAMP during the first 10 hr of starvation. The pools in 39 of the mutants remained low and relatively static during this period. However, amoebae of one mutant, strain HC151, exhibited significantly elevated levels of intracellular cAMP during vegetative growth and for several hours after starvation. A more detailed analysis of this mutant indicated that the elevated cAMP pools in these cells are a consequence of the premature appearance and partial activation of an adenylate cyclase. The mutation(s) altering adenylate cyclase regulation in this strain appears to map in linkage group IV. Complementation tests between strain HC151 and another mutant, HH201, which has recently been shown to produce an adenylate cyclase activity precociously [1], indicated that the mutations affecting adenylate cyclase activity in these strains map at different loci. Although both of these mutations behave recessively in heterozygous diploids with respect to gross development, an examination of early cAMP metabolism and terminal spore differentiation in these diploids suggest that these mutations are at least partially expressed during some stage(s) of the developmental cycle.     

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.     

Suppression of defective RAS1 and RAS2 functions in yeast by an adenylate cyclase activated by a single amino acid change.

We have constructed the yeast strain TS1, with the RAS2 gene replaced by mutant allele encoding a partially defective gene product, and with an inactive RAS1 gene. TS1 cells accumulate as unbudded cells upon temperature shift from 30 to 37 degrees C, thus showing that the RAS1 and RAS2 gene functions are important for progression through the G1 phase of the cell cycle. After the isolation of revertants able to grow at the nonpermissive temperature, we have found that a chromosomal point mutation can bypass the G1 arrest of TS1 and cdc25 cells, and the lethality of ras1 ras2 mutants. The mutation predicts the replacement of threonine by isoleucine at position 1651 of yeast adenylate cyclase. The RAS-independent, as well as the RAS-dependent adenylate cyclase activity, is increased by the mutation. Like the wild-type enzyme, the RAS-dependent activity of the mutant adenylate cyclase is turned on by the GTP-bound form of the RAS2 protein. The amino acid sequence surrounding the threonine 1651 shows similarity with protein kinase substrates. Possible implications for the function of adenylate cyclase are discussed.     

Evidence for adenylate cyclase as a scaffold protein for Ras2–Ira interaction in Saccharomyces cerevisie

Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.     

Responsiveness to Exogenous Camp of a Saccharomyces Cerevisiae Strain Conferred by Naturally Occurring Alleles of Pde1 and Pde2

The Saccharomyces cerevisiae 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 PDE1 and PDE2, 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 CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae 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.     

Deletion of Snf1 Affects the Nutrient Response of Yeast and Resembles Mutations Which Activate the Adenylate Cyclase Pathway

We have isolated a snf1/ccr1 mutant of Saccharomyces cerevisiae which loses viability upon starvation and fails to accumulate glycogen in response to abrupt depletion of phosphate or glucose. A snf1 null mutant is sensitive to heat stress and starvation and fails to accumulate glycogen during growth in rich medium. The phenotypes of the snf1 mutants are those commonly associated with an overactivation of the adenylate cyclase pathway. Mutations in adenylate cyclase or RAS2 which decrease the level of cAMP in the cell moderate the snf1 phenotype. In contrast, a mutation in RAS2 (RAS2val19) which increases the level of cAMP or a mutation in the regulatory subunit (BCY1) of cAMP-dependent protein kinase which results in unregulated cAMP-dependent protein kinase activity accentuates the snf1 phenotype. However, the action of SNF1 in the stress response appears at least partly independent of cAMP-dependent protein kinase because a snf1 phenotype is observed in a strain that lacks all three of the genes that encode the catalytic subunits of cAMP-dependent protein kinase. SNF1 therefore acts at least in part through a cAMP-independent pathway.     

Yeast cdc35 mutants are defective in adenylate cyclase and are allelic with cyr1 mutants while CAS1, a new gene, is involved in the regulation of adenylate cyclase

Newly isolated temperature-sensitive cdc35 mutants of Saccharomyces cerevisiae have been characterized. They show the morphology, growth and conjugation characteristics typical of class-A or class-II start mutants. The cdc35 mutation induces a significant decrease of the intracellular cAMP level and produces a thermolabile adenylate cyclase. By classical genetic criteria the CDC35 gene is identical with the structural gene of adenylate cyclase, CYR1. The results of the mutant selection, the kinetics of macromolecule accumulation and the cell-density change of cdc35 mutants at the restrictive temperature, indicate that CDC35 function may not be cell cycle-specific. A new mutation, cas1, was isolated and partially characterized. It mediates the suppression by external cAMP of the unlinked cdc35 mutation. It causes a slight increase of the intracellular cAMP level and has strong effects on the adenylate cyclase activities, especially on the Mg2+ dependent activity. The data suggest that the CAS1 protein is a controlling element of adenylated cyclase. The CAS1 locus is different from the RAS1 and RAS2 loci.     

Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.     

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.     

Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down-regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhibitory effects of GTP gamma S and GDP beta S on cAMP binding are reduced; the stimulatory effect of cAMP on GTP gamma S binding is lost in fgd A mutants. (d) Basal high-affinity GTPase activity is reduced 40% and the stimulatory effect of cAMP is decreased from 40% in wild type to 30% in fgd A. (e) GTP-mediated stimulation and inhibition of adenylate cyclase is normal in mutant membranes. The results suggest a defective interaction between cell surface cAMP receptors and a specific G-protein in fgd A mutants. This interaction appears to be essential for nearly all signal transduction pathways in Dictyostelium discoideum.     

Modulation of expression of the stress-inducible p118 of Saccharomyces cerevisiae by cAMP. II. A study of p118 expression in mutants of the cAMP cascade

In the preceding paper, we have identified a protein of Mr = 118,000 which is induced by stress conditions that lead to cessation of DNA synthesis and cell division (Verma, R., Iida, H., and Pardee, A.B. (1988) J. Biol. Chem. 263, 8569-8575). In the current study, we have investigated the possible role this protein may play in cellular proliferation by studying p118 expression in mutants of the cAMP metabolic pathway. The cyr 1-2 mutant gene encodes a thermolabile adenylate cyclase whose activity is only 7% of wild type even at permissive temperatures (23 degrees C). We have found that at 23 degrees C, the G1 period was 5-fold longer in cyr 1-2 than in CYR1+ cells and that p118 was constitutively expressed in these slow cycling mutants. Addition of 8-bromo-cAMP to cyr 1-2 mutants restored growth at both the restrictive and permissive temperatures and resulted in a shut-off in the synthesis of p118. The effect of the analog on p118 expression was rapid, preceding the increase in cell number and percentage-budded cells. In contrast to wild type cells, p118 synthesis was not induced by sulfur starvation in RAS2val19 mutants possessing high levels of adenylate cyclase activity and bcy1 mutants defective in the regulatory subunit of cAMP-dependent protein kinase. A large body of evidence exists supporting a role of cAMP in positive control of cell proliferation. It is therefore possible that conditions which decrease cAMP arrest growth through a chain of events that include p118 induction.     

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.     

Multiple regulation of the activity of adenylate cyclase in Escherichia coli

Summary We have studied the correlation between the activities of adenylate cyclase (ATP pyrophosphatelyase-(cyclizing); EC 4.6.1.1) and in vivo rates of synthesis and intracellular concentrations of adenosine 3,5 cyclic monophosphate (cAMP) under various growth conditions in wild-type Escherichia coli and in mutants lacking or overproducing the cAMP receptor protein (CAP). We showed that when wild-type bacteria are grown in the presence of a variety of carbon sources the intracellular concentrations of cAMP are inversely related to the adenylate cyclase activities determined in permeabilized cells, suggesting that the carbon source-dependent modulation of cAMP levels is not directly related to the regulation of adenylate cyclase activity. In mutants lacking functional CAP (crp) the in vivo rates of cAMP synthesis are several hundred-fold higher than in the wild-type parent without a parallel increase of adenylate cyclase activities. In a strain carrying multiple copies of the crp gene and overproducing CAP the activity of adenylate cyclase is severely inhibited, although the in vivo rate of cAMP synthesis is similar to the parental strain. We interpret these results as indicating that CAP controls mainly the activity rather than the synthesis of adenylate cyclase.     

A new extragenic suppressor of cya mutation. Mutant cyclic AMP receptor protein with an increased affinity for cyclic AMP.

A strain bearing an extragenic suppressor of cya mutation was isolated as a second-site revertant of an adenylate cyclase deficient strain. The mutant was unable to synthesize cAMP but showed normal fermentation profiles and growth properties on a variety of carbon sources. The site of reversion was mapped in, or near, the structural gene for the cAMP receptor protein. Structural alteration of the protein was directly demonstrated by the following biochemical observations: (i) A 10-fold decrease in the dissociation constant for cAMP, (ii) an acidic shift in the isoelectric point, and (iii) the altered binding properties to lambdah80dlac ps DNA.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to adenylate cyclase.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.     

Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.     

Role of a guanine nucleotide binding protein, G alpha 2, in regulation of adenylate cyclase in Dictyostelium discoideum

Two substances, cAMP and 2,3-dimercapto-1-propanol (BAL) are known to induce transient activation of adenylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) has a deletion in a gene for G alpha 2, a guanine nucleotide binding protein and cannot activate the cyclase in response to cAMP. We found that BAL induced activation in the frigid mutant. This result suggests that the BAL-induced activation is independent of G alpha 2 and that BAL mimics a role of activated G alpha 2. We also found that cAMP promoted the BAL-induced activation. This result suggests that cAMP plays a role in activation through a mechanism in which G alpha 2 is not involved. We lastly showed that continuous cAMP stimulation could not inhibit the BAL-induced activation in the frigid mutant. Since the cAMP-induced inhibition observed in the wild type strain (NC4) proceeds with the time course identical to the cAMP-induced adaptation (Oyama, submitted), this result suggests that G alpha 2 is involved in adaptation of adenylate cyclase.