A second stress-inducible glutathione S-transferase gene from Schizosaccharomyces pombe

A second glutathione S-transferase gene (GST II) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined contains 1908 bp including an open reading frame of 230 amino acids that would encode a protein of a molecular mass of 26843.4 Da. The amino acid sequence of the putative GST II is very homologous with that of the previously isolated GST gene (GST I) located in the same chromosome III of S. pombe. The cloned GST II gene produces the functional GST in S. pombe, and it gives much higher GST in the stationary phase than in the exponential phase. Regulation of the GST II gene was studied using the GST II-lacZ fusion. The synthesis of beta-galactosidase from the fusion plasmid is greatly enhanced by the treatments with oxidative stresses such as menadione and mercuric chloride. It is also induced by o-dinitrobenzene, one of the GST substrates. NO-generating S-nitroso-N-acetylpenicillamine has a weak induction effect on the expression of GST II gene. These results indicate that the S. pombe GST II gene is involved in the oxidative stress response and detoxification. However, physiological meaning on the existence of the two similar GST genes in S. pombe remains unknown yet.     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

Characterization and regulation of the gamma-glutamyl transpeptidase gene from the fission yeast Schizosaccharomyces pombe

The structural gene for the putative gamma-glutamyl transpeptidase (GGT) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined sequence contained 3324 bp and encoded the predicted 630 amino acid sequence of GGT, which resembles counterparts in Homo sapiens, Rattus norvegicus, Saccharomyces cerevisiae, and Escherichia coli. The S. pombe cells harboring the cloned GGT gene showed about twofold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGT gene was functional. To monitor the expression of the S. pombe GGT gene, we fused the fragment 1085 bp upstream of the cloned GGT gene into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pGT98. The synthesis of beta-galactosidase from the fusion plasmid in S. pombe cells was enhanced by treatments with NO-generating sodium nitroprusside (SN), L-buthionine-(S,R)-sulfoximine (BSO), and glycerol. The GGT mRNA level in the S. pombe cells was increased by SN and BSO. Involvement of Pap1 in the induction of the GGT gene by SN and BSO was observed.     

Pap1-mediated regulation of thioredoxin gene from Schizosaccharomyces pombe

The genomic DNA encoding thioredoxin (TRX) was previously isolated from the fission yeast Schizosaccharomyces pombe. In this investigation, regulation of the S. pombe TRX gene was studied in lacZ translational fusions. The synthesis of beta-galactosidase from the fusion plasmid pYKT24 was significantly enhanced by treatments with cadmium chloride, zinc chloride, and high temperatures. Synthesis of beta-galactosidase from the fusion plasmid was significantly decreased by higher concentrations (5 microM, 10 microM) of mercuric chloride, whereas it was enhanced by its lower concentration (1 microM). Diamide affected the synthesis of beta-galactosidase in the same manner with mercuric chloride. However, high osmolarity had no effect on the beta-galactosidase synthesis from the fusion plasmid pYKT24. Various fusion plasmids were constructed to carry serially deleted upstream regions of the TRX gene. Pap1 mediates the regulation of the S. pombe TRX gene. The upstream region, between 987 and 1,270 bp from the translational initiation point, is responsible for the regulation.     

Stress-dependent regulation of a monothiol glutaredoxin gene from Schizosaccharomyces pombe

Glutaredoxin (Grx) is a small, heat-stable protein acting as a multi-functional glutathione-dependent disulfide oxidoreductase. In this work, a gene encoding the monothiol glutaredoxin Grx4 was cloned from the genomic DNA of the fission yeast Schizosaccharomyces pombe. The determined DNA sequence carries 1706 bp, which is able to encode the putative 244 amino acid sequence of Grx with 27 099 Da. It does not contain an intron, and the sequence CGFS is found in the active site. Grx activity was increased 1.46-fold in S. pombe cells harboring the cloned Grx4 gene, indicating that the Grx4 gene is in vivo functioning. Although aluminum, cadmium, and hydrogen peroxide marginally enhanced the synthesis of beta-galactosidase from the Grx4-lacZ fusion gene, NO-generating sodium nitroprusside (0.5 mmol/L and 1.0 mmol/L) and potassium chloride (0.2 mol/L and 0.5 mol/L) significantly enhanced it. The Grx4 mRNA level was also enhanced after the treatment with sodium nitroprusside and potassium chloride. The synthesis of beta-galactosidase from the Grx4-lacZ gene was increased by fermentable carbon sources, such as glucose (lower than 2%) and sucrose, but not by nonfermentable carbon sources such as acetate and ethanol. The basal expression of the S. pombe Grx4 gene did not depend on the presence of Pap1. These results imply that the S. pombe monothiol Grx4 gene is genuinely functional and regulated by a variety of stresses.     

Characterization,expression and regulation of a third gene encoding glutathione S-transferase from the fission yeast

A third gene encoding glutathione S-transferase (GSTIII) was cloned from the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined was found to contain 2110 base pairs including an open reading frame of 242 amino acids that would encode a protein of a molecular mass of 26,620 Da. The cloned GSTIII gene could be expressed in S. pombe, S. cerevisiae and Escherichia coli cells which gave 1.4-, 2.1-, and 3.0-fold higher GST activity in an assay using 1-chloro-2,4-dinitrobenzene as a substrate, respectively. The cloned GSTIII gene caused higher survivals of S. pombe cells on solid media with cadmium chloride or mercuric chloride. The GSTIII protein has 16% and 18% homologies with the GSTI and GSTII proteins, respectively. To independently monitor the regulation of the GSTIII gene, its 1168 bp upstream region and N-terminal 33 amino acid-coding region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357. The synthesis of beta-galactosidase from the fusion plasmid pGY357 was greatly enhanced by cadmium chloride (50 microM), cupric chloride (10 microM), aluminum chloride (5 mM, 10 mM), mercuric chloride (1 microM), and zinc chloride (10 mM). However, the synthesis of beta-galactosidase from the fusion plasmid pGY357 was not affected by superoxide-generating menadione, and o-dinitrobenzene, whereas they could significantly induce the expression of the GSTI and GSTII genes of S. pombe. The overproduced Pap1 inhibited the induction of beta-galactosidase synthesis from the fusion plasmid pGY357 by cadmium chloride, which is opposite to the previously known role of Pap1 in the response to oxidative stress. Our results collectively indicate that the three GST genes of S. pombe are subjected to different regulatory mechanisms. The major role of the GSTIII protein in S. pombe may be the detoxification of various metals.     

Characterization of a second gene encoding gamma-glutamyl transpeptidase from Schizosaccharomyces pombe

The first gene encoding gamma-glutamyl transpeptidase (GGTI) of the fission yeast has previously been characterized, and its expression was found to be regulated by various oxidative stress-inducing agents. In this work, a second gene, encoding GGTII, was cloned and characterized from the fission yeast Schizosaccharomyces pombe. The structural gene encoding GGTII was amplified from the genomic DNA of the fission yeast and ligated into the shuttle vector pRS316 to generate the recombinant plasmid pPHJ02. The determined sequence contains 3040 bp and is able to encode the putative 611 amino acid sequence of GGTII, which resembles the counterparts of Saccharomyces cerevisiae, Homo sapiens, Rattus norvegicus, and Escherichia coli. The DNA sequence also contains 940-bp upstream and 289-bp downstream regions of the GGTII gene. The Schizosaccharomyces pombe cells harboring plasmid pPHJ02 showed about 4-fold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGTII gene is functional. The S. pombe cells containing the cloned GGTII gene were found to contain higher levels of both intracellular glutathione (GSH) content and GSH uptake. The S. pombe cells harboring plasmid pPHJ02 showed increased survival on solid media containing hydrogen peroxide, diethylmaleate, aluminum chloride, cadmium chloride, or mercuric chloride. The GGTII mRNA level was significantly elevated by treatment with GSH-depleting diethylmaleate. These results imply that the S. pombe GGTII gene produces functional GGTII protein and is involved in the response to oxidative stresses in S. pombe cells.     

Cloning, expression and regulation of Schizosaccharomyces pombe gene encoding thioltransferase

The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.     

Characterization of Schizosaccharomyces pombe RNA triphosphatase

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the β–γ phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast Schizosaccharomyces pombe. Pct1p hydrolyzes the γ phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and P_i in the presence of manganese or cobalt (K_m = 19 µM ATP; k_cat = 67 s^–1). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I_0.5 = 1 mM), pyrophosphate (I_0.5 = 0.4 mM) and tripolyphosphate (I_0.5 = 30 µM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.     

Characterization of two protein kinases from Schizosaccharomyces pombe involved in the regulation of DNA repair.

We have identified two novel genes designated hhp1+ and hhp2+ in the fission yeast Schizosaccharomyces pombe. The hhp1+ and hhp2+ genes encode two closely related protein kinases that share significant sequence identities with Hrr25p from Saccharomyces cerevisiae. Characterization of strains harboring single and double mutations in the hhp+ genes reveals DNA repair defects in these cells. Schizosaccharomyces pombe strains lacking either or both Hhp activities reveal differences in their ability to withstand DNA lesions caused by either methyl methanesulfonate (MMS) or gamma-rays which correlate with their ability to repair DNA strand breaks caused by these agents. We suggest that Hhp1 and Hhp2 are involved in the regulation of distinct and overlapping DNA repair pathways in S. pombe.     

Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe

The Schizosaccharomyces pombe cwg2⁺ gene encodes the β-subunit of geranylgeranyl transferase I (GGTase I), which participates in the post-translational C-terminal modification of several small GTPases, allowing their targeting to the membrane. Using the two-hybrid system, we have identified the cwp1⁺ gene that encodes the α-subunit of the GGTase I. cwp1p interaction with cwg2p was mapped to amino acids 1–244 or 137–294 but was not restricted to amino acids 137–244. The genomic cwp1⁺ was isolated and sequenced. It has two putative open reading frames of 677 and 218 bp, separated by a 51 bp intron. The predicted amino acid sequence shows significant similarity to GGTase I α-subunits from different species. However, complementation of Saccharomyces cerevisiae ram2-1 mutant by overexpressing the cwp1⁺ gene was not possible. Expression of both cwg2⁺ and cwp1⁺ in Escherichia coli allowed ‘in vitro’ reconstitution of the GGTase I activity. S. pombe cells expressing the mutant enzyme containing the cwg2-1 mutation do not grow at 37°C, but the growth defect can be suppressed by the addition of sorbitol. Actin immunostaining of the cwg2-1 mutant strain grown at 37°C showed an abnormal distribution of actin patches. The cwg2-1 mutation was identified as a guanine to adenine substitution at nucleotide 604 of the coding region, originating the change A202T in the cwg2p. Deletion of the cwg2 gene is lethal; Δcwg2 spores can divide two or three times before losing viability. Most cells have aberrant morphology and septation defects. Overexpression of the rho1G15VC199R double-mutant allele in S. pombe caused loss of polarity but was not lethal and did not render the (1–3)β-D -glucan synthase activity independent of GTP. Therefore, geranylgeranylation of rho1p is required for the appropriate function of this GTPase.     

Characterization of O-mannosyltransferase family in Schizosaccharomyces pombe

Protein O-glycosylation is an essential protein modification in eukaryotic cells. In Saccharomyces cerevisiae, O-mannosylation is initiated in the lumen of the endoplasmic reticulum by O-mannosyltransferase gene products (Pmt1p-7p). A search of the Schizosaccharomyces pombe genome database revealed a total of three O-glycoside mannosyltransferase homologs (ogm1+, ogm2+, and ogm4+), closely related to Saccharomyces cerevisiae PMT1, PMT2, and PMT4. Although individual ogm genes were not found to be essential, ogm1Delta and ogm4Delta mutants exhibited aberrant morphology and failed to agglutinate during mating. The phenotypes of the ogm4Delta mutant were not complemented by overexpression of ogm1+ or ogm2+, suggesting that each of the Ogm proteins does not have overlapping functions. Heterologous expression of a chitinase from S. cerevisiae in the ogm mutants revealed that O-glycosylation of chitinase had decreased in ogm1Delta cells. A GFP-tagged Fus1p from S. cerevisiae was specifically not glycosylated and accumulated in the Golgi in ogm4Delta cells. These results indicate that O-glycosylation initiated by Ogm proteins plays crucial physiological roles and can serve as a sorting determinant for protein transport of membrane glycoproteins in S. pombe.