Phosphorylation of the GTS1 gene product of the yeast Saccharomyces cerevisiae and its effect on heat tolerance and flocculation

The GTS1 gene from the yeast Saccharomyces cerevisiae showed pleiotropic effects on yeast phenotypes, including an increase of heat tolerance in stationary-phase cells and an induction of flocculation. Here, we found that the GTS1 product, Gts1p, was partially phosphorylated at some serine residue(s) in cells grown on glucose. Studies using mutants of protein kinase A (PKA) and CDC25, the Ras-GTP exchange activator, showed that PKA positively regulated the phosphorylation level of Gts1p. Overexpression of Gts1p in a mutant with attenuated PKA activity did not show any increase of heat tolerance and partially decreased flocculation inducibility, suggesting that phosphorylation of Gts1p is required for induction of these phenomena.     

PSK2 coordinates glucose metabolism and utilization to maintain ultradian clock-coupled respiratory oscillation in Saccharomyces cerevisiae yeast

Ultradian clock-coupled respiratory oscillation (UCRO) in an aerobic continuous culture of Saccharomyces cerevisiae S288C is principally regulated by control of certain redox reactions of energy metabolism. It is also modulated by the metabolism of storage carbohydrates during adaptation to environmental change. However, the mechanism of cell sensing and response to environmental nutrients in UCRO is unknown. The purpose of the present study was to determine the role of PSK2 kinase in UCRO in yeast. S. cerevisiae in culture showed oscillation in PSK2 mRNA levels with a definite phase relationship to the respiratory oscillation. Furthermore, inactivation of Psk2 by gene disruption severely affected UCRO and its decline to undetectable levels within 2 days. In addition, the extracellular and intracellular glucose concentrations of PSK2 deletion mutants in culture were higher and lower, respectively, than those of the wild type. PSK2 mutant cells showed no alteration in redox state. Furthermore, the levels of storage carbohydrates such as glycogen and trehalose fluctuated in PSK2 mutants with attenuated amplitudes comparable to those in the wild type. The results indicated that PSK2 kinase is important for the uptake of glucose and regulation of storage-carbohydrate synthesis and hence the maintenance of an unperturbed continuously oscillating state.     

Generation and maintenance of synchrony in Saccharomyces cerevisiae continuous culture

Cultures of Saccharomyces cerevisiae grown continuously produce an autonomous oscillation in many metabolic outputs. The most conveniently measured variable, i.e., dissolved oxygen concentration, oscillates with a period of 40-55 min. Previously we have identified two compounds capable of resetting phase, acetaldehyde and hydrogen sulfide. The phase-response curves constructed for acetaldehyde show a strong (Type 0) response at 3.0 mM and a weak (Type 1) response at 1.0 mM. Ammonium sulfide phase-response curves (pulse injected at 1.0 microM and 3.0 microM) revealed that sulfide is only an effective perturbation agent when endogenous sulfide concentrations are at a maximum. Also only Type 1 phase responses were observed. When the phase-response curve for sulfite (at 3.0 M) was constructed, phase responses were at a maximum at 60 degrees, indicating the possible involvement of sulfite in cell synchronization. It is concluded that endogenously produced acetaldehyde and sulfite tune the oscillation of mitochondrial energization state whereas sulfide mediates population synchrony.     

Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae

Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the β-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation. The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps. The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level. The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.     

Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae

Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the β-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation. The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps. The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level. The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.     

Severe reduction of superoxide dismutase activity in the yeast Saccharomyces cerevisae with the deletion or overexpression of GTS1

We report herein that the level of reactive oxygen species (ROS) observed using dihydrorhodamine is much higher in either GTS1-deleted (gts1Delta) or GTS1-overexpressing (TMpGTS1) transformants than in the wild-type and that the levels of protein carbonyls are increased and the glutathione levels are decreased in both transformants. Consistently, the activities of superoxide dismutases (SODs) in both gts1Delta and TMpGTS1 were severely weakened, while the protein levels of both Cu/Zn-SOD and Mn-SOD were not so changed. As the intracellular copper levels were significantly increased in both transformants, we hypothesized that, in either gts1Delta or TMpGTS1 cells, the imbalanced homeostasis of copper induced an accumulation of ROS which caused inactivation of SODs further increasing ROS levels.     

Interaction of the GTS1 gene product with glyceraldehyde- 3-phosphate dehydrogenase 1 required for the maintenance of the metabolic oscillations of the yeast Saccharomyces cerevisiae.

We previously reported that GTS1 is involved in regulating ultradian oscillations of the glycolytic pathway induced by cyanide in cell suspensions as well as oscillations of energy metabolism in aerobic continuous cultures. Here, we screened a yeast cDNA library for proteins that bind to Gts1p using the yeast two-hybrid system and cloned multiple TDH cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the zinc-finger and dimerization sites of Gts1p were required for full ability to bind GAPDH, and Gts1ps mutated at these sites lost the ability to regulate both aerobic and unaerobic ultradian oscillations of energy metabolism. Of the three TDH genes, only TDH1 fluctuated at the mRNA level in continuous culture and its deletion resulted in the disappearance of the oscillation without any affect on growth rate. This loss of biological rhythms in the TDH1-deleted mutant was rescued by the expression of TDH1 but not of TDH2 or TDH3 under the control of the TDH1 promoter. Thus, we hypothesized that Gts1p plays a role in the regulation of metabolic oscillation by interacting with the TDH1 product, GAPDH1, in yeast.     

Deletion of OSH3 gene confers resistance against ISP-1 in Saccharomyces cerevisiae

Sphingolipids have been reported to regulate the growth and death of mammalian and yeast cells, but their precise mechanisms are unknown. In this paper, it was shown that the deletion of the oxysterol binding protein homologue 3 (OSH3) gene confers hyper resistance against ISP-1, an inhibitor of sphingolipid biosynthesis, in the yeast Saccharomyces cerevisiae. Furthermore, the overexpression of the ROK1 gene, which directly binds to Osh3p, conferred resistance against ISP-1, and the deletion of the KEM1 gene, which regulates microtubule functions, exhibited ISP-1 hypersensitivity. And yet, an ISP-1 treatment caused an abnormal mitotic spindle formation, and the ISP-1-induced cell cycle arrest was rescued by the deletion of the OSH3 gene. Taken together, it is suggested that the expression levels of the OSH3 gene influence the ISP-1 sensitivity of S. cerevisiae, and the sphingolipids are necessary for normal mitotic spindle formation in which the Osh3p may play a pivotal role.     

The ubiquitin ligase SCF(Grr1) is required for Gal2p degradation in the yeast Saccharomyces cerevisiae

F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCF(Grr1), including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCF(Grr1)-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1Delta mutant cells imply that Gal2p is not the primary target of SCF(Grr1) ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.     

The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae.

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.     

The KNS1 gene of Saccharomyces cerevisiae encodes a nonessential protein kinase homologue that is distantly related to members of the CDC28/cdc2 gene family

Summary A novel protein kinase homologue (KNS1) has been identified in Saccharomyces cerevisiae. KNS1 contains an open reading frame of 720 codons. The carboxy-terminal portion of the predicted protein sequence is similar to that of many other protein kinases, exhibiting 36% identity to the cdc2 gene product of Schizosaccharomyces pombe and 34% identity to the CDC28 gene product of S. cerevisiae. Deletion mutations were constructed in the KNS1 gene. kns1 mutants grow at the same rate as wild-type cells using several different carbon sources. They mate at normal efficiencies, and they sporulate successfully. No defects were found in entry into or exit from stationary phase. Thus, the KNS1 gene is not essential for cell growth and a variety of other cellular processes in yeast.     

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae. Received: 20 November 1996 / Accepted: 25 February 1997     

The paromomycin resistance mutation (parr-454) in the 15 S rRNA gene of the yeast Saccharomyces cerevisiae is involved in ribosomal frameshifting

Summary The leaky expression of the yeast mitochondrial geneoxi1, containing a frameshift mutation (+1), is caused by natural frameshift suppression, as shown previously (Fox and Weiss-Brummer 1980). A drastic decrease in the natural level of frameshifting is found in the presence of thepar ^r-454 mutation, localized at the 3′ end of the 15 S rRNA gene. This mutation causes resistance to the antibiotic paronomycin in the yeast strains D273-10B and KL14-4A (Li et al. 1982; Tabak et al. 1982). The results of this study imply that in the yeast strain 777-3A this mutation alone is sufficient for restriction of the level of natural frameshifting but is insufficient to confer resistance to paromomycin. A second mutation, arising spontaneously with a frequency of 10⁻⁴ leads, in combination with thepar ^r-454 mutation, to full paromomycin resistance in strain 777-3A.