Interaction of cAMP with the CDC25-mediated step in the cell cycle of budding yeast

Addition of exogenous cAMP to cultures of the start mutant cdc25-1 of Saccharomyces cerevisiae shifted to restrictive temperature causes a partial reversion of the mutated phenotype, with a marked increase of the percentage of budded cells. This effect is coupled to a progression in the cell cycle, as demonstrated by DNA histograms obtained by flow cytometry. Moreover cdc25 cells have a high intracellular cAMP content also at restrictive temperature, and no change in the cAMP content was seen during a transition from restrictive to permissive temperature. These data suggest that CDC25 gene product allows cell proliferation by interacting with a cAMP-mediated mechanism.     

Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae.

The cell division cycle of the yeast Saccharomyces cerevisiae is triggered at the stage called 'START'. Many results strongly suggest that adenylate cyclase is an essential element of the control of START. We report here results arguing for a positive control of the cAMP level by the CDC25 gene, another gene of START. Firstly, cdc25 cells can be rescued by extracellular cAMP. Secondly, the cellular cAMP content drops when thermosensitive cdc25 mutant cells are shifted to restrictive temperature. We report the molecular cloning of the CDC25 gene by complementation of cdc25 mutant cells. The identity of the cloned gene was confirmed by site-specific gene re-integration experiments and segregation analysis: the isolated fragment is shown to integrate into the cdc25 gene locus. When transferred in cdc25 mutant cells this DNA prevents the drop of the cAMP level at restrictive temperature. This gene is transcribed in a 5200-nucleotides mRNA. We have determined the nucleotide sequence of a 5548-bp DNA fragment which shows an uninterrupted open reading frame (ORF) coding for a 1587-amino acid polypeptide chain. Only the C-terminal part of the ORF appears to be essential for the complementation of the cdc25-5 allele, suggesting a multidomain protein.     

Glucose-induced activation of plasma membrane H(+)-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.

Addition of glucose-related fermentable sugars or protonophores to derepressed cells of the yeast Saccharomyces cerevisiae causes a 3- to 4-fold activation of the plasma membrane H(+)-ATPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP synthesis, incubation at the restrictive temperature reduced the extent of H(+)-ATPase activation. Incubation of non-temperature-sensitive strains, however, at such temperatures also caused reduction of H(+)-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H(+)-ATPase activation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H(+)-ATPase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAMP-protein kinase A signaling pathway is not required for glucose activation of the H(+)-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phosphorylating enzymes hexokinase PI and PII and glucokinase showed that activation of the H(+)-ATPase with glucose or fructose was completely dependent on the presence of a kinase able to phosphorylate the sugar. These and other data concerning the role of initial sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H(+)-ATPase have a common initiation point.     

Isolation and nucleotide sequence of a Saccharomyces cerevisiae protein kinase gene suppressing the cell cycle start mutation cdc25

We have isolated two unlinked yeast genes complementing the cell division cycle mutant cdc25-1, one containing the wild type allele CDC25 and the other acting as an extragenic suppressor of the cdc25-1 lesion if present on a multicopy plasmid. Nucleotide sequence analysis of the suppressor gene has revealed an open reading frame that encodes a 45,000-dalton protein belonging to the protein kinase family. The cdc25-suppressing protein kinase (PK-25) shows 48% sequence similarity to the catalytic subunit (CA) of mammalian cAMP-dependent protein kinase and 27-31% similarity to cyclic nucleotide-independent enzymes, including the yeast CDC28 gene product. The PK-25 gene was targeted by integrative transformation into a chromosomal region unlinked to the CYR2 site, the structural gene of CA. The cdc25-suppressing protein kinase is also functionally different from CA, since cyr2 strains deficient in the free catalytic subunit remain temperature sensitive if transformed with a multicopy plasmid containing the PK-25 gene. Furthermore, a deficiency of the cAMP-binding regulatory subunit (RA) caused by the bcy1 mutation fails to suppress the cdc25 mutation, indicating that PK-25 does not interact with the cAMP receptor protein. Our data suggest that the cdc25 suppressor gene encodes a cAMP-independent protein kinase involved in the control of the cell cycle start.     

The cdc30 mutation in Saccharomyces cerevisiae results in a temperature-sensitive isoenzyme of phosphoglucose isomerase

The cdc30 mutation in the yeast Saccharomyces cerevisiae causes cell cycle arrest late in nuclear division when cells are shifted from the permissive temperature of 25 degrees C to the restrictive temperature of 36.5 degrees C. Cell cycle arrest at 36.5 degrees C is dependent upon the carbon source used: a shift-up in glucose containing media results in cell cycle blockade, whereas a shift-up in ethanol, fructose, glycerol, glycerol plus ethanol, or mannose does not. Metabolite analyses showed accumulation of glucose 6-phosphate in a cdc30-bearing strain after a temperature shift-up in glucose-containing medium. Thermal denaturation studies and kinetic measurements indicate the existence of two isoenzymes of phosphoglucose isomerase (EC 5.3.1.9); one of which is apparently altered in the temperature-sensitive cell cycle mutant. We propose that the gene products of both the CDC30 and PG11 genes are required for cell cycle progression in glucose media and that the PGI1 gene product has a regulatory function over the CDC30 gene product.     

Control of the cAMP pathway by the cell cycle start function, CDC25, in Saccharomyces cerevisiae

We investigated the relationship in Saccharomyces cerevisiae between the cell cycle start function, CDC25, and two mutants defining components of the cAMP pathway. The thermolabile adenylate cyclase mutant cyr1-2(ts) is phenotypically similar to the temperature-sensitive mutant cdc25(ts) in that both mutants, when shifted to the restrictive temperature, arrest in G1 of the cell cycle and permit the initiation of meiosis and sporulation. The mutant bcy1 [a lesion resulting in a low level of regulatory (R) subunit and a high level of active, catalytic (C) subunit of the cAMP-dependent protein kinase] suppresses the temperature-sensitive phenotype of cyr1-2(ts) and confers an asporogenous phenotype. We found that cdc25(ts) complemented cyr1-2(ts), and, unlike cyr1-2(ts), was not suppressible by bcy1, demonstrating that CYR1 and CDC25 must encode different functions. Also our results indicate that CDC25 does not encode the R subunit of the cAMP-dependent protein kinase. In addition, although the cdc25(ts)bcy1 double mutant was temperature sensitive like cdc25(ts), we found that the cdc25(ts)bcy1 homozygous diploid was asporogenous like bcy1/bcy1. The inability of the cdc25(ts)bcy1 double mutant to sporulate demonstrated that CDC25 does not encode the C subunit of the cAMP kinase, and indicated that the CDC25 function modulates the cAMP pathway to control meiosis and sporulation. Further, the temperature-sensitive phenotype of the double mutant, and hence the inability of bcy1 to suppress cdc25(ts), suggested that a second CDC25 cell cycle function exists which is independent of the cAMP pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.     

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.     

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.     

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.     

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.     

Cyclic AMP controls the plasma membrane H+-ATPase activity from Saccharomyces cerevisiae

The thermosensitive G1-arrested cdc35-10 mutant from Saccharomyces cerevisiae, defective in adenylate cyclase activity, was shifted to restrictive temperature. After 1 h incubation at this temperature, the plasma membrane H+-ATPase activity of cdc35-10 was reduced to 50%, whereas that in mitochondria doubled. Similar data were obtained with cdc25, another thermosensitive G1-arrested mutant modified in the cAMP pathway. In contrast, the ATPase activities of the G1-arrested mutant cdc19, defective in pyruvate kinase, were not affected after 2 h incubation at restrictive temperature. In the double mutants cdc35-10 cas1 and cdc25 cas1, addition of extracellular cAMP prevented the modifications of ATPase activities observed in the single mutants cdc35-10 and cdc25. These data indicate that cAMP acts as a positive effector on the H+-ATPase activity of plasma membranes and as a negative effector on that of mitochondria.     

The Saccharomyces cerevisiae start mutant carrying the cdc25 mutation is defective in activation of plasma membrane ATPase by glucose.

Activation of plasma membrane ATPase by the addition of glucose was examined in several cell division cycle mutants of Saccharomyces cerevisiae. The start mutant carrying the cdc25 mutation was shown to be defective in ATPase activation at the restrictive temperature. Genetic analysis showed that lack of growth and defective activation of ATPase at the restrictive temperature were caused by the same mutation. It was also found that CDC25 does not map at the same locus as the structural gene of plasma membrane ATPase (PMA1). We conclude that the product of CDC25 controls the activation of ATPase.     

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

Chromosome Separation and Exit from Mitosis in Budding Yeast: Dependence on Growth Revealed by cAMP-Mediated Inhibition

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.     

The high-affinity glucose uptake system is not required for induction of the RAS-mediated cAMP signal by glucose in cells of the yeast Saccharomyces cerevisiae

Addition of glucose or related fermentable sugars to yeast cells grown on non-fermentable carbon sources, triggers a RAS-protein mediated cAMP signal which induces a protein phosphorylation cascade. The high-affinity glucose uptake system in yeast cells is known to be glucose-repressible and only functional in strains containing at least one active kinase. In strains containing point or disruption mutations in the SNF3 gene, which codes for the high-affinity glucose carrier, the glucose-induced cAMP signal is still present. This indicates that the previously demonstrated requirement of a functional kinase for the induction of the cAMP signal, does not reflect requirement of high-affinity sugar transport. It also indicates that the unknown glucose-repressible protein in the induction sequence of the RAS-mediated cAMP signal is not the high-affinity sugar carrier.     

Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats.

We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitive [corrected] defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras. In contrast to the yeast RAS genes, Xenopus N-ras rescues the cdc15 mutation. Moreover, overexpression of Xenopus N-ras in S. cerevisiae does not activate the RAS-cyclic AMP (cAMP) pathway; rather, it results in decreased levels of intracellular cAMP in both mutant cdc15 and wild-type cells. Furthermore, we show that lowering cAMP levels is sufficient to allow cells with a nonfunctional Cdc15 protein to complete the mitotic cycle. These results suggest that a key step of the cell cycle is dependent upon a phosphorylation event catalyzed by cAMP-dependent protein kinase. The second clone, beta TrCP (beta-transducin repeat-containing protein), encodes a protein of 518 amino acids that shows significant homology to the beta subunits of G proteins in its C-terminal half. In this region, beta Trcp is composed of seven beta-transducin repeats. beta TrCP is not a functional homolog of S. cerevisiae CDC20, a cell cycle gene that also contains beta-transducin repeats and suppresses the cdc15 mutation.