In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.     

Identification of the structural gene and nonsense alleles for adenylate cyclase in Saccharomyces cerevisiae.

Tetraploid strains of Saccharomyces cerevisiae carrying different dosages of the CYR1+ gene have been constructed. Adenylate cyclase activity observed in these tetraploid strains was proportional to the dosage of the active CYR1+ gene. Of the 57 mutants requiring adenosine 3',5'-monophosphate for growth at 35 degrees C, two allelic temperature-sensitive cyr1 mutants produced thermolabile adenylate cyclase. Crude extract and plasma membrane fraction of cyr1 mutant cells had no adenylate cyclase activity when assayed with GTP or 5'-guanylyl imidodiphosphate in the presence of Mn2+ or Mg2+. Plasma membrane and Lubrol-soluble plasma membrane fractions obtained from the temperature-sensitive cyr1 mutant were thermolabile compared with those from the wild-type strain. Three cyr1 mutants carried nonsense mutations susceptible to ochre (UAA) suppressors, SUP3 and SUP-o, and had no detectable level of adenylate cyclase activity. It is concluded that the cyr1 mutants carry lesions in the structural gene for adenylate cyclase.     

A guanine nucleotide-sensitive adenylate cyclase in the yeast Saccharomyces cerevisiae

Adenylate cyclase in particulate extracts of Saccharomyces cerevisiae utilized either MnATP or MgATP as substrate. A mutation in the CYR1 gene, which codes for the catalytic unit of yeast adenylate cyclase (Matsumoto, K., Uno, I., and Ishikawa, T. (1983) Cell 32, 417-423), eliminated utilization of both MgATP and MnATP, indicating that a single enzyme was responsible for both activities. GTP and guanylyl-5'-imidodiphosphate stimulated yeast adenylate cyclase, while a GDP analog, guanosine-5'-O-(2-thiodiphosphate), competitively inhibited this stimulation. Thermal inactivation studies distinguished putative guanine-nucleotide regulatory protein (N) from the catalytic unit (C) of yeast adenylate cyclase. Yeast N, which conferred guanine nucleotide regulation and the ability to utilize MgATP on yeast C, was quickly inactivated by incubation of particulate extracts at 30 degrees C. In contrast, yeast C, which apparently utilized MnATP as substrate in the absence of a functional N protein, resisted inactivation at 30 degrees C. These observations suggested that physically distinct protein components mediated the catalytic activity of yeast adenylate cyclase and its regulation by guanine nucleotides. These findings indicate a striking homology between the adenylate cyclase systems of S. cerevisiae and those of vertebrate cells.     

Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae

The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP. The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio. In addition 'in vitro' mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role.     

The functional domain of adenylate cyclase associated with entry into meiosis in Saccharomyces cerevisiae.

Diploid yeast cells that carry a part of the CYR1 gene deficient in a region coding for the N-terminal domain of adenylate cyclase were growth arrested and accumulated unbudded cells after inoculation into complete medium or nitrogen-free medium, but produced many cells which had one or more buds after incubation in sporulation medium. The cells incubated in sporulation medium had abnormal spindles which were free from the spindle pole bodies, larger in size, or frequently distributed in cytoplasm. The levels of cyclic AMP in these cells did not decrease to the wild-type level after transfer to the sporulation medium and remained at a constant level. The results suggest that the N-terminal domain of adenylate cyclase is associated with the regulatory function for sporulation. The environmental signals for sporulation may be transferred to the adenylate cyclase system through a factor that negatively interacts with the N-terminal domain of this enzyme.     

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

Starvation-associated mutagenesis in yeast Saccharomyces cerevisiae is affected by Ras2/cAMP signaling pathway

The number of revertants with restored ability to form colony increases in a time-dependent manner during long-term selective starvation of dense mutant microbial cultures. This is due to starvation-associated (also called adaptive) mutations that arise in a replication independent manner. Here we report that in Saccharomyces cerevisiae the frequency of starvation-associated reversions of mutant genes whose products are necessary for amino acids biosynthesis are influenced by Ras2/cAMP signaling pathway. This signaling pathway is a yeast general regulatory pathway involved in nutritional sensing, UV response, sporulation control and life span control and its changes are manifested in both, cell cycle and life cycle. Inactivation of the RAS2 gene causes an increase in number of starvation-associated revertants in comparison to an isogenic wild type strain and a strain with constitutively activated Ras2/cAMP signaling pathway. Therefore, we suggest that starvation-associated mutagenesis is different from spontaneous mutagenesis and is related to the cellular capacity to adopt distinct physiological states in response to environmental signals.     

Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.     

Properties and possible functions of the adenylate cyclase in plasma membranes of Saccharomyces cerevisiae

We have examined the possible role of adenosine 3',5'-phosphate (cAMP) in functions associated with the plasma membranes of Saccharomyces cerevisiae. Purified membranes from this source contained an adenylate cyclase which was insensitive to activation by fluoride or guanine nucleotides, only weakly responsive to changes of carbon source in the growth medium, and strongly stimulated by vanadate. They also contained at least two classes of receptor proteins for guanine nucleotides (as measured by binding of labeled 5'-guanylyl methylene diphosphate) with apparent dissociation constants equal to 1.0 x 10(-7) and 3 x 10(-6) M, a protein kinase capable of phosphorylating added histones, the activity of which was stimulated by cAMP, and cAMP receptors that may function as regulatory subunits for this kinase. Membrane proteins were also susceptible to phosphorylation by endogenous kinase(s), with polypeptides of apparent molecular weights equal to 160 x 10(3), 135 x 10(3), 114 x 10(3), and 58 x 10(3) as the major targets. Of these, the 114,000-molecular-weight polypeptide was probably identical to the proton-translocating ATPase of the membranes. However, the cAMP-dependent protein kinase did not appear to be involved in these reactions. Intact (rho+ or rho0) cells responded to dissipation of the proton electrochemical gradient across their plasma membranes by rapid and transient changes in their intracellular level of cAMP, as suggested earlier (J. M. Trevillyan and M. L. Pall, J. Bacteriol., 138:397-403, 1979). Thus, although yeast plasma membranes contain all the essential components of a stimulus-responsive adenylate cyclase system, the precise nature of the coupling device and the targets involved remain to be established.     

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.     

The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway

The gene corresponding to the S. cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins. Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity. The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene. These results suggest that normal RAS is dependent on CDC25 function. Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein. We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.     

The genetic control of cell growth and development in yeast Saccharomyces cerevisiae. Disturbed sporulation in diploids with decreased activity of the Ras/cAMP signal transduction pathway

Seven haploid strains (four with the MAT alpha mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast. Previous phenotypic analysis assigned six of these strains to a physiological group of strains with a lower activity of the Ras/cAMP signal transduction pathway. The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media. Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency). The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway. All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34 degrees C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose). Sporulation proved to depend on temperature and medium composition. The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.     

A putative second adenylate kinase-encoding gene from the yeast Saccharomyces cerevisiae.

Sequencing of the region upstream from the yeast RAD3 gene has revealed an open reading frame (ORF) of 225 amino acids (aa) that could encode a 25.3-kDa polypeptide. The predicted aa sequence of this ORF is homologous with that of several eukaryotic adenylate kinase (Adk)-encoding genes, including the yeast gene, ADK1. These findings suggest that the yeast Saccharomyces cerevisiae has a second Adk-encoding gene, tentatively designated as ADK2.     

An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations.

Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.     

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product.

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.