A novel cis-acting cysteine-responsive regulatory element of the gene for the high-affinity glutathione transporter of Saccharomyces cerevisiae.

We cloned a DNA fragment from Saccharomyces cerevisiae that complemented the deficiency in high-affinity glutathione transport activity conferred by a gsh11 mutation, and found that the ORF responsible was YJL212c, which had already been designated as OPT1 and HGT1 by others. Northern analysis clearly demonstrated that this ORF, now referred to as OPT1/ HGT1/ GSH11, was induced by sulfur starvation and repressed by adding cysteine to the growth medium. Reporter gene assays showed that a segment spanning the region between positions -371 and -355 was essential for the regulation of this gene. A sequence of 9 nt, CCGCCACAC (from -364 to -356), in this region was shown to be required for protein binding, using an electrophoretic mobility shift assay. Based on these results, we propose that CCGCCACAC comprises the core of a cis-acting element involved in cysteine-responsive gene regulation in S. cerevisiae.

Saccharomyces cerevisiae cultured under aerobic and anaerobic conditions: air-level oxygen stress and protection against stress.

Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells. Aerobiosis increased both the compounds and enzyme activities. The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine beta-synthase.

Role of the sulphate assimilation pathway in utilization of glutathione as a sulphur source by Saccharomyces cerevisiae.

Mutants unable to grow on medium containing glutathione as a sole source of sulphur (GSH medium) were isolated from Saccharomyces cerevisiae strains carrying met17(deficiency of O-acetylserine and O-acetylhomoserine sulphydrylase). They were defective in the high-affinity glutathione transport system, GSH-P1. Newly acquired mutations belonged to the same complementation group, gsh11. However, it became apparent that gsh11 conferred the mutant phenotype not by itself but in collaboration with met17. Moreover, mutations conferring the defect in sulphate assimilation made the cell unable to grow on GSH medium in collaboration with gsh11. From this finding, we propose that the sulphate assimilation pathway acts as a sulphur-recycling system and that this function is especially vital to the cell when the supply of glutathione is limited.

Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation.

Using a Saccharomyces cerevisiae strain having the activities of serine O-acetyl-transferase (SATase), O-acetylserine/O-acetylhomoserine sulphydrylase (OAS/OAH SHLase), cystathionine beta-synthase (beta-CTSase) and cystathionine gamma-lyase (gamma-CTLase), we individually disrupted CYS3(coding for gamma-CTLase) and CYS4 (coding for beta-CTSase). The obtained gene disruptants were cysteine-dependent and incorporated the radioactivity of (35)S-sulphate into homocysteine but not into cysteine or glutathione. We concluded, therefore, that SATase and OAS/OAH SHLase do not constitute a cysteine biosynthetic pathway and that cysteine is synthesized exclusively through the pathway constituted with beta-CTSase and gamma-CTLase; note that OAS/OAH SHLase supplies homocysteine to this pathway by acting as OAH SHLase. From further investigation upon the cys3-disruptant, we obtained results consistent with our earlier suggestion that cysteine and OAS play central roles in the regulation of sulphate assimilation. In addition, we found that sulphate transport activity was not induced at all in the cys4-disruptant, suggesting that CYS4 plays a role in the regulation of sulphate assimilation.

S-(1,2-Dicarboxyethyl)glutathione in yeast: partial purification of its synthesizing enzyme.

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) was found in Saccharomyces cerevisiae, but not in bacterial species nor in a unicellular alga (Acetabularia acetabulum). The enzyme that catalyzes condensation of L-malate and glutathione (GSH) to form DCE-GS was partially purified from baker's yeast. It had a molecular mass of 49 kDa and was monomeric and the Km values were 2.2 and 1.4 mM for L-malate and GSH, respectively. The enzyme had a pH optimum of 7.5. DCE-GS levels in yeast cells were significantly higher in aerobic cultures than in anaerobic ones. DCE-GS was synthesized in cells cultured between 20 and 35 degrees C.

Glutathione transport systems of the budding yeast Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae was shown to have two kinetically distinguishable glutathione transport systems. While one with high affinity (GSH-P1; KT = 0.045 mM) was regulated, the other with low affinity (GSH-P2; KT > 2 mM) was not. GSH-P1 was highly specific to glutathione, and its activity was quickly lost by suspending the cells in buffer solutions. This activity loss was not observed if glucose-containing buffer was used. In addition, rho-isolates had only about one half of the glutathione transport activity of the original (rho+) strain. Therefore, it is concluded that GSH-P1 is an ATP-driven transport system. Strong and moderate inhibition of GSH-P1 by protonophores and ionophores, respectively, are attributed to competition for ATP between GSH-P1 and proton- and cation-pumps, respectively.

Regulation of sulphate assimilation in Saccharomyces cerevisiae.

We examined how the activity of O-acetylserine and O-acetylhomoserine sulphydrylase (OAS/OAH) SHLase of Saccharomyces cerevisiae is affected by sulphur source added to the growth medium and genetic background of the strain. In a wild-type strain, the activity was repressed if methionine, cysteine or glutathione was added to the growth medium. However, in a strain deficient of cystathionine gamma-lyase, cysteine and glutathione were repressive, but methionine was not. In strains deficient of serine O-acetyltransferase (SATase), OAS/OAH SHLase activity was low regardless of sulphur source and was further lowered by cysteine and glutathione, but not by methionine. From these observations, we concluded that S-adenosylmethionine should be excluded from being the effector for regulation of OAS/OAH SHLase. Instead, we suspected that S. cerevisiae would have the same regulatory system as Escherichia coli for sulphate assimilation; i.e. cysteine inhibits SATase to lower the cellular concentration of OAS which is required for induction of the sulphate assimilation enzymes including OAS/OAH SHLase. Subsequently, we obtained data supporting this speculation.

HAM1, the gene controlling 6-N-hydroxylaminopurine sensitivity and mutagenesis in the yeast Saccharomyces cerevisiae.

The ham1 mutant of yeast Saccharomyces cerevisiae is sensitive to the mutagenic and lethal effects of the base analog, 6-N-hydroxylaminopurine (HAP). We have isolated a clone from a centromere-plasmid-based genomic library complementing HAP sensitivity of the ham1 strain. After subcloning, a 3.4 kb functional fragment was sequenced. It contained three open reading frames (ORFs) corresponding to proteins 353, 197 and 184 amino acids long. LEU2+ disruptions of the promoter and N-terminal part of the gene coding 197 amino acids long protein led to moderate and strong sensitivity to HAP, respectively, and were allelic to the original ham1-1 mutation. Thus this ORF represents the HAM1 gene. The deduced amino acid sequence of HAM1 protein was not similar to any protein sequence of the SwissProt database. The HAM1 gene was localized on the right arm of chromosome X between cdc8 and cdc11. Spontaneous mutagenesis was not affected by the ham1::LEU2 disruption mutation.

Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+].

The yeast non-Mendelian factor [psi+] has been suggested to be a self-modified protein analogous to mammalian prions. Here it is reported that an intermediate amount of the chaperone protein Hsp104 was required for the propagation of the [psi+] factor. Over-production or inactivation of Hsp104 caused the loss of [psi+]. These results suggest that chaperone proteins play a role in prion-like phenomena, and that a certain level of chaperone expression can cure cells of prions without affecting viability. This may lead to antiprion treatments that involve the alteration of chaperone amounts or activity.

Mutagenicity of 5-bromouracil and N6-hydroxyadenine studied by yeast oligonucleotide transformation assay.

The mutagenicity of 5-bromouracil (BrU) and N6-hydroxyadenine (HA) was tested by means of the yeast oligonucleotide transformation procedure. BrU-containing oligonucleotide was not mutagenic; although two mutants (per 200 micrograms oligonucleotide) were obtained, they were attributed to base insertion or base substitution at positions different from BrU. This result supports the view that BrU mutagenesis is dependent on intracellular nucleotide pool imbalance. In contrast, HA-containing oligonucleotide was highly mutagenic; 56 mutants (per 140 micrograms oligonucleotide) were obtained. Of 21 induced mutants examined, 20 had G and one had C at the HA position, a result indicating that HA-->G changes took place. To provide back-up evidence, we carried out a general reversion assay for base HA using a set of yeast tester strains, and the results showed that HA induces exclusively AT-to-GC and GC-to-AT transitions. We conclude that in S. cerevisiae HA is a classic base analog mutagen, causing AT-to-GC and GC-to-AT transitions by ambiguous base pairing. The present work has clearly demonstrated the usefulness of the oligonucleotide transformation procedure for elucidating mutagenicity of modified bases.

The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae.

Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.

Purification and properties of Saccharomyces cerevisiae cystathionine beta-synthase.

Cystathionine beta-synthase (beta-CTSase), which catalyses cystathionine synthesis from serine and homocysteine, was purified to homogeneity from Saccharomyces cerevisiae. The molecular mass of the enzyme was estimated to be 235 kDa by gel filtration and 55 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that it is a homotetramer. The N-terminal amino acid sequence of the enzyme perfectly coincided with that deduced from the nucleotide sequence of CYS4, except for the absence of initiation The purified beta-CTSase catalysed cysteine synthesis from serine (or O-acetylserine) and H2S. From this finding, we discuss the multifunctional nature and evolutionary divergence of S-metabolizing enzymes.

Asd-homothallism of Saccharomyces cerevisiae: identification of asd1-1 as an allele of sir4 and detection of alpha-specific suppressors of it.

Asd-homothallism of Saccharomyces cerevisiae involves a life cycle characterized by a non-mating phenotype and endomitotic diploidization. The former trait is determined by a single mutation, asd1-1. This mutation was mapped between hom2 and lys4 on the right arm of chromosome IV and was complemented by the cloned SIR4 gene. Therefore, we conclude that asd1-1 is an allele of sir4-11 and renamed it sir4-11. Endomitotic diploidization of asd-homothallism is caused by the collaboration of three to four mutations including sir4-11. In the course of this study, we detected alpha-specific suppressors of sir4-11.

Relationship between chromosomal alpha-specific suppressors of sir4-11 and polymorphism of the HMRa-bearing fragment.

In the course of studying sir4-11 which is responsible for the non-mating phenotype of asd-homothallism of Saccharomyces cerevisiae, we detected chromosomal alpha-specific suppressors of it. By examining the mating-type cassette constitution of two strains and the spore-clones derived from a diploid culture formed by a mass mating between these strains, we obtained the following results; (1) the HMRa-bearing HindIII-Bg/II restriction fragment was dimorphic, as judged by size, within each tetrad, (2) while one form was common to all tetrads, the other varied among tetrads, (3) spore-clones with the alpha-specific suppressor of sir4-11 had the variant forms while those without had the common form, and (4) while the two parents had the common form, each independent diploid clone had the common form plus a variant form. From these results, we conclude that the mating of cells in certain combinations induces a change of DNA structure at or near HMRa in a mating-pair specific manner and that the change makes HMRa non-derepressible or non-functional when derepressed.

Cystathionine gamma-lyase of Saccharomyces cerevisiae: structural gene and cystathionine gamma-synthase activity.

Purification of Saccharomyces cerevisiae cystathionine gamma-lyase (gamma-CTLase) was hampered by the presence of a protein migrating very close to it in various types of column chromatography. The enzyme and the contaminant were nevertheless separated by polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that they are coded for by CYS3 (CYI1) and MET17 (MET25), respectively, leading to the conclusion that CYS3 is the structural gene for gamma-CTLase and that the contaminant is O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase). Based on these findings, we purified gamma-CTLase by the following strategy: (1) extraction of OAS/OAH SHLase from a CYS3-disrupted strain; (2) preparation of antiserum against it; (3) identification of a strain devoid of the OAS/OAH SHLase protein using this antiserum; and (4) extraction of gamma-CTLase from this strain. Purified gamma-CTLase had cystathionine gamma-synthase (gamma-CTSase) activity if O-succinylhomoserine, but not O-acetylhomoserine, was used as substrate. From this notion we discuss the evolutional relationship between S. cerevisiae gamma-CTLase and Escherichia coli gamma-CTSase.

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae: regulatory roles of methionine and S-adenosylmethionine reassessed.

cys4-1, a mutation in the reverse trans-sulphuration pathway, relieves the sulphate assimilation pathway and homocysteine synthase from methionine-mediated repression. Since the mutation blocks the synthesis of cysteine from methionine downstream from homocysteine, this indicates that neither methionine nor S-adenosylmethionine serve as low-molecular-mass effectors in this regulatory system, contradicting earlier hypotheses.