The large N-terminal domain of Cdc25 protein of the yeast Saccharomyces cerevisiae is required for glucose-induced Ras2 activation

The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor for Ras proteins whose catalytic domain is highly homologous to Ras-guanine nucleotide exchange factors from higher eukaryotes. In this study, glucose-induced Ras activation and cAMP response were investigated in mutants lacking the N-terminal domain of Cdc25 or where the entire CDC25 coding sequence was substituted by an expression cassette for a mammalian guanine nucleotide exchange factor catalytic domain. Our results suggest that an unregulated, low Ras guanine nucleotide exchange factor activity allows a normal glucose-induced cAMP signal that appears to be mediated mainly by the Gpr1/Gpa2 system, but it was not enough to sustain the glucose-induced increase of Ras2-GTP normally observed in a wild-type strain.     

The a-factor pheromone of Saccharomyces cerevisiae is essential for mating.

The Saccharomyces cerevisiae pheromone a-factor is produced by a cells and interacts with alpha cells to cause cell cycle arrest and other physiological responses associated with mating. Two a-factor structural genes, MFA1 and MFA2, have been previously cloned with synthetic probes based on the a-factor amino acid sequence (A. Brake, C. Brenner, R. Najarian, P. Laybourn, and J. Merryweather, cited in M.-J. Gething [ed.], Protein transport and secretion, 1985). We have examined the function of these genes in a-factor production and mating by construction and analysis of chromosomal null mutations. mfa1 and mfa2 single mutants each exhibited approximately half the wild-type level of a-factor activity and were proficient in mating, whereas the mfa1 mfa2 double mutant produced no a-factor and was unable to mate. These results demonstrate that both genes are functional, that each gene makes an equivalent contribution to the a-factor activity and mating capacity of a cells, and that a-factor plays an essential role in mating. Strikingly, exogenous a-factor did not alleviate the mating defect of the double mutant, suggesting that an a cell must be producing a-factor to be an effective mating partner.     

Cdc15 is required for spore morphogenesis independently of Cdc14 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-DeltaNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.     

The Cdc6 protein is ubiquitinated in vivo for proteolysis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of pre-replicative complexes that are required for firing DNA replication at origins at the beginning of S phase. Cdc6p protein levels oscillate during the cell cycle. In a normal cell cycle the presence of this protein is restricted to G1, partly because the CDC6 gene is transcribed only during G1 and partly because the Cdc6p protein is rapidly degraded at late G1/early S phase. We report here that the Cdc6p protein is degraded in a Cdc4-dependent manner, suggesting that phosphorylated Cdc6 is specifically recognized by the ubiquitin-mediated proteolysis machinery. Indeed, we have found that Cdc6 is ubiquitinated in vivo and degraded by a Cdc4-dependent mechanism. Our data, together with previous observations regarding Cdc6 stability, suggest that under physiological conditions budding yeast cells degrade ubiquitinated Cdc6 every cell cycle at the beginning of S phase.     

Glucose-induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein

Functional mapping of the cell cycle START gene CDC25 has revealed two domains which are dispensable for viability (germination and growth in glucose media), but are essential for sporulation and differentially involved in glucose-induced cAMP signaling. The transient rise of cAMP is completely prevented by various deletions within the amino-terminal half (alpha domain) of the CDC25 gene product. In contrast, the deletion of the carboxy-terminal 38 residues (beta 2 domain) results in a rapid, but persisting, rise of cAMP. Our data suggest that the alpha domain of the CDC25 protein is involved in glucose signal transduction, whereas the beta 2 domain is required for downregulating the cAMP control chain.     

dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.     

The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae.

Glucose uptake mutants have not been previously obtained in Saccharomyces cerevisiae, possibly because there seem to be at least two transport systems, of low and high affinities. We showed that snf3 (sucrose nonfermenting) mutants did not express high-affinity glucose uptake. Furthermore, their growth was completely impaired on low concentrations of glucose in the presence of antimycin A (which blocks respiration). Several genes which complemented the original snf3 gene were obtained on multicopy plasmids. Some of them, as well as plasmid-carried SNF3 itself, conferred a substantial increase in high-affinity glucose uptake in both snf3 and wild-type hosts. The effects of glucose on the expression of such a plasmid-determined high-affinity uptake resembled those in the wild type. Other genes complementing snf3 seemed to cause an increase in low-affinity glucose uptake. We suggest that SNF3 may function specifically in high-affinity glucose uptake, which is needed under some conditions of growth on low glucose concentrations. SNF3 itself or the other complementing genes may specify components of the glucose uptake system.     

Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity.

Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.     

The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes.     

High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control.

Saccharomyces cerevisiae mutants defective in growth on low glucose concentration (lgn mutants) were isolated and screened for abnormal glucose transport. Nine complementation groups were identified, falling into two broad groups: those unable to significantly derepress high-affinity (low-Km) glucose uptake (lgn1, lgn4, lgn5, lgn7, and lgn8), and those with elevated repressed levels of high-affinity uptake that either derepress to normal or near normal levels of high-affinity uptake with loss of low-affinity transport (lgn2 and lgn3) or derepress only slightly, appearing to have an intermediate yet constitutive level of high-affinity transport (lgn6 and lgn9). Further analysis of the lgn mutations revealed pleiotropic phenotypes most consistent with the true defect being in regulation or expression of glucose repression and derepression. The kinetics of glucose uptake in strains carrying known mutations preventing derepression of glucose-repressible functions (snf1, snf2, snf4, and snf6) demonstrated that three of these mutations (snf1, snf4, and snf6) were similarly defective in derepression of high-affinity glucose uptake. The snf2 and snf5 mutations had no apparent effect on glucose uptake. Two mutations resulting in constitutive expression of glucose-repressible functions, cid1 and reg1, resulted in constitutive expression of high-affinity glucose uptake. These data support the conclusion that high-affinity glucose uptake in Saccharomyces cerevisiae is under general glucose repression control. The implications of other properties of these mutants are discussed.     

The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function.

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.     

Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose.

By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations. These previous determinations had led to the proposal that S. cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter. We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity for glucose changes. We conclude that the transporter or group of transporters is constitutive and that regulation of glucose transport occurs via a factor that modifies the affinity of the transporters and not via the synthesis of different kinetically independent transporters. Such a mechanism could, for instance, be accommodated by the binding of kinases causing a change in affinity for glucose.     

Individual messenger RNA half lives in Saccharomyces cerevisiae.

Summary We have measured the decay half-life of functional messenger RNA (mRNA) for some thirty different proteins in the yeast Saccharomyces cerevisiae. Production of newly synthesized mRNA was halted by raising the temperature of a culture of a temperature-sensitive mutant, ts 136. Aliquots of this culture were pulsed-labelled with [³⁵S]-methionine at various times after the temperature shift and the radioactive proteins separated on the two-dimensional gel electrophoresis system of O'Farrell. We find a range in the decay half lives of individual mRNA species which varies from 3.5 min to greater than 70 min. We find three general classes of decay curves, (a) simple exponential (first order); some of these showed a shoulder before onset of exponential decay; (b) bi-component or multi-component concave upward; (c) initial stimulation of rate of mRNA synthesis, followed by virtually undetectable decay.     

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.     

The suppressor gene scl1+ of Saccharomyces cerevisiae is essential for growth

In Saccharomyces cerevisiae, the SCL-1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, crl3 [McCusker and Haber, Genetics 119 (1988a) 303-315]. The wild-type scl1+ gene was isolated by screening subclones of the 35-kb region between TRP5 and LEU1 for restoration of the ts phenotype in an SCL1-1 crl3-2 strain. The scl1+ mRNA is about 900 nt long and encodes an open reading frame of 810 bp. The polypeptide deduced from scl1+ possesses a putative secretory signal peptide. The 5'-noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF. Gene disruption of scl1+ demonstrates that it is an essential gene.     

The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.

We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.     

Physical interaction of Cdc28 with Cdc37 in Saccharomyces cerevisiae

The Cdc37 protein in Saccharomyces cerevisiae is thought to be a kinase-targeting subunit of the chaperone Hsp90. In a genetic screen, four protein kinases were identified as interacting with Cdc37 - Cdc5, Cdc7, Cdc15 and Cak1. This result underlines the importance of Cdc37 for the folding of protein kinases. In addition, we showed that Ydj1, a yeast DnaJ homolog belonging to the Hsp40 family of chaperones, genetically interacts with Cdc37. No physical interaction has so far been detected between Cdc37 and Cdc28, although genetic interactions (synthetic lethality and mutation suppression), and biochemical studies have suggested that these two proteins functionally interact. We found that, when separately expressed, the N-terminal lobe of Cdc28 interacted strongly with the C-terminal moiety of Cdc37 in a two-hybrid system. This was not the case for the full-length Cdc28 protein. We present models to explain these results.