Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae

Ono, Bun-ichiro; Tanaka, Kouji; Naito, Kazuhide; Heike, Chinatsu; Shinoda, Sumió; Yamamoto, Satoshi; Ohmori, Shinji; Oshima, Takao; Toh‐e, Akio

doi:10.1128/jb.174.10.3339-3347.1992

Cited by 46 publications

(32 citation statements)

References 40 publications

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“…Downregulated genes were identified as CLB2, CLN3, CYS3, and SMF2, with CYS3 being amplified with several different primer combinations. Clb2 is a G 2 -specific B-type cyclin (46), Cln3 is a G 1 cyclin (38), and Cys3 is a cystathionine gamma-lyase which catalyzes the biosynthesis of cysteine (35). The function of Smf2 is unknown.…”

Section: Resultsmentioning

confidence: 99%

CLN1 and Its Repression by Xbp1 Are Important for Efficient Sporulation in Budding Yeast

Mai

Breeden

2000

Molecular and Cellular Biology

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Xbp1, a transcriptional repressor of Saccharomyces cerevisiae with homology to Swi4 and Mbp1, is induced by stress and starvation during the mitotic cycle. It is also induced late in the meiotic cycle. Using RNA differential display, we find that genes encoding three cyclins (CLN1, CLN3, and CLB2), CYS3, and SMF2 are downregulated when Xbp1 is overexpressed and that Xbp1 can bind to sequences in their promoters. During meiosis, XBP1 is highly induced and its mRNA appears at the same time as DIT1 mRNA, but its expression remains high for up to 24 h. As such, it represents a new class of meiosis-specific genes. Xbp1-deficient cells are capable of forming viable gametes, although ascus formation is delayed by several hours. Furthermore, Xbp1 target genes are normally repressed late in meiosis, and loss of XBP1 results in their derepression. Interestingly, we find that a deletion of CLN1 also reduces the efficiency of sporulation and delays the meiotic program but that sporulation in a ⌬cln1 ⌬xbp1 strain is not further delayed. Thus, CLN1 may be Xbp1's primary target in meiotic cells. We hypothesize that CLN1 plays a role early in the meiotic program but must be repressed, by Xbp1, at later stages to promote efficient sporulation.

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Section: Resultsmentioning

confidence: 99%

CLN1 and Its Repression by Xbp1 Are Important for Efficient Sporulation in Budding Yeast

Mai

Breeden

2000

Molecular and Cellular Biology

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“…A bacterial expression vector, pTrc99A (Pharmacia), was digested with NcoI and PstI so that it could accept a pGEMCYS3-derived fragment (nucleotide 14-686) which encoded the N-terminal segment of the CYS3. Nucleotide 1 designates the A of the initiator methionine codon as described previously by Ono et al (39). The complementary synthetic oligonucleotides (Rikaken Co. Ltd.) encoding the missing NH2-terminal five amino acids were used as an adaptor (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Content of the sulfhydryl group was determined by the methods of Sakai (41) and of Elmann (10) (Fig. 2) (39), the deduced amino acid sequence of CYS3 has highly significant homology with several enzymes involved in metabolism of sulfur-containing amino acids; E. coli CT 1y-synthase (8), ClT l-lyase (9), rat CIT ly-lyase (11), and S. cerevisiae OAS-OAH sulfhydrylase (6,24). All these proteins are known to have oligomeric structures composed of identical subunits of molecular weights ranging from 40,000 to 50,000 and to require PLP as a cofactor (11,17,44,47).…”

Section: Methodsmentioning

confidence: 99%

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Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein

et al. 1993

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By screening a yeast genomic library, we isolated and characterized a gene rescuing the cysteine requirement in a "cys1" strain of Saccharomyces cerevisiae. Except for four residues in the open reading frame composed of 1,182 nucleotides, the DNA sequence was the same as that for the CYS3 (CYI1) gene, encoding cystathionine gamma-lyase (EC 4.4.1.1), and isolated previously as a cycloheximide-induced gene (B. Ono, K. Tanaka, K. Naito, C. Heike, S. Shinoda, S. Yamamoto, S. Ohmori, T. Oshima, and A. Toh-e, J. Bacteriol. 174:pp.3339-3347, 1992). S. cerevisiae "cys1" strains carry two closely linked mutations; one (cys1) causes a defect in serine O-acetyltransferase (EC 2.3.1.30), and another, designated cys3, impairs cystathionine gamma-lyase activity. Rescue of the cysteine requirement by the gene encoding cystathionine gamma-lyase is consistent with both defects being responsible for the cysteine auxotrophy. In an effort to further determine the physicochemical and enzymatic properties of this enzyme, a coding fragment was cloned into an Escherichia coli expression plasmid, and the protein was produced in the bacteria. The induced protein was extracted by sonication and purified to homogeneity through one course of DEAE-cellulose column chromatography. The yield of the protein was approximately 150 mg from cells cultured in 1 liter of L broth. The protein showed molecular weights of approximately 194,000 and 48,000 (for the subunit), suggesting a tetrameric structure. An s20,w value of 8.8 was estimated by centrifugation in a sucrose concentration gradient. No sulfhydryl groups were detected, which is consistent with the absence of cysteine residues in the coding sequence. The isoelectric point was at pH 5.2. The protein showed a number of cystathionine-related activities, i.e., cystathionine beta-lyase (EC 4.4.1.8), cystathionine gamma-lyase, and cystathionine gamma-synthase (EC 4.2.99.9) with L-homoserine as substrate. In addition, we demonstrated L-homoserine sulfhydrylase (adding H2S) activity but could find no detectable serine O-acteyltransferease activity. In this paper, we compare the enzymatic properties of the protein with those of homologous enzymes previously reported and discuss the possibility that this enzyme has a physiological role as cystathionine Beta-lyase and cystathionine gamma-synthase in addition to its previously described role as cystathionine gamma-lyase.

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“…Since alliinase is supposed to be a vacuolar protein [3], it needs a signal peptide [15]; ASA, A. sativum alliinase [15]; ATCBL, Arabidopsis thaliana cystathionine β-lyase [41]; CGS-1, A. thaliana cystathionine γ-synthase [42]; CYS-3, S. cerevisiae cystathionine γ-lyase [43]; MET17, S. cerevisiae cystathionine γ-synthase [45]; METC, S. cerevisiae cystathionine β-lyase [46]; METZ, Pseudomonas aeruginosa O-succinyl homoserine sulfhydrylase [47]; MGL, P. putida methionine γ-lyase [48].…”

Section: Molecular Comparison With Alliinases From Other Plantsmentioning

confidence: 99%

Alliinase [S‐alk(en)yl‐ L‐cysteine sulfoxide lyase] from Allium tuberosum (Chinese chive)

Manabe

Hasumi

Sugiyama

et al. 1998

European Journal of Biochemistry

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Alliinase [S-alk(en)yl-L-cysteine sulfoxide lyase], a pyridoxal-phosphate-(Pxy-P)-dependent enzyme, is responsible for the degradative conversion of S-alk(en)yl-L-cysteine sulfoxide to volatile odorous sulfurcontaining metabolites in Allium plants. We have purified alliinase from shoots of Allium tuberosum (Chinese chive) to apparent homogeneity by SDS/polyacrylamide gel electrophoresis. A cDNA clone encoding alliinase was isolated from a cDNA library constructed from whole plants of A. tuberosum by hybridization screening with a synthetic 50-residue oligonucleotide encoding a conserved region of the alliinases from onion and garlic. The isolated cDNA encoded a protein of 476 amino acid residues with a molecular mass of 54 083 Da. The deduced amino acid sequence exhibited 66Ϫ69% identities with those of reported alliinases from onion, garlic and shallot. The partial amino acid sequence, which was determined for a V8 protease-digested peptide fragment of the purified alliinase, was perfectly matched with the sequence deduced from the cDNA. An expression vector of recombinant alliinase cDNA was constructed in yeast. The catalytically active protein was in the soluble fraction of transformed yeast. Site-directed mutagenesis experiments indicated that Lys280 was essential for the catalytic activity and, thus, a possible Pxy-P-binding residue. The mRNA expression of the alliinase gene comprising a multigene family in the shoots of green plants was twofold higher than that in the roots of green plants ; however, the expression in the shoots of etiolated plants was only 13% that in green shoots, although the expression in the roots was not remarkably different between in green and etiolated plants. Immunohistochemical investigation indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A. tuberosum.Keywords : Allium; alliinase ; cDNA cloning; pyridoxal 5′-phosphate; sulfur metabolism.The plants belonging to genus Allium generally produce characteristic odorous sulfur-containing compounds. These volatile sulfur compounds are formed from non-volatile S-alk(en)yl-L-cysteine sulfoxide (ACS), as a precursor, through enzymatic degradation, which is catalyzed by so-called alliinase [S-alk-(en)yl-L-cysteine sulfoxide lyase] [1, 2]. In onion (Allium cepa) cells, alliinase is assumed to be localized in vacuoles, thereby being separated from ACS present in the cytosol [3]. Upon rupture of the cells, the enzyme can react with the substrate to yield sulfenic acid as the first sulfur-containing volatile compound (Scheme 1). Numerous sulfur-containing metabolites, such as thiosulfinates, thiols, disulfides, etc. are secondarily produced from sulfenic acid through non-enzymatic reactions [4]. These

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Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae

Cited by 46 publications

References 40 publications

CLN1 and Its Repression by Xbp1 Are Important for Efficient Sporulation in Budding Yeast

CLN1 and Its Repression by Xbp1 Are Important for Efficient Sporulation in Budding Yeast

Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein

Alliinase [S‐alk(en)yl‐ L‐cysteine sulfoxide lyase] from Allium tuberosum (Chinese chive)

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