• Journal of Microbiology and Biotechnology
• /
• v.24 no.9
• /
• pp.1207-1215
• /
• 2014

Candida albicans, the major human fungal pathogen, undergoes morphological transition from the budding yeast form to filamentous growth in response to nitrogen starvation. In this study, we identified a new function of GST2, whose expression was required for filamentous growth of C. albicans under nitrogen-limiting conditions. The Gst2p showed Gst activity and required response to oxidative stress. The ${\Delta}gst2$ mutant displayed predominantly yeast phase growth in low ammonium media. Such morphological defect of ${\Delta}gst2$ mutants was not rescued by overexpression of Mep2p, Cph1p, or Efg1p, but was rescued by either overexpression of a hyperactive $RAS1^{G13V}$ allele or through exogenous addition of cyclic AMP. In addition, the ${\Delta}gst2$ mutants had lower levels of RAS1 transcripts than wild-type cells under conditions of nitrogen starvation. These results were consistent with the Ras1-cAMP pathway as a possible downstream target of Gst2p. These findings suggest that Gst2p is a significant component of nitrogen starvation-induced filamentation in C. albicans.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2002.10a
• /
• pp.135-139
• /
• 2002

Yeast cells respond to condition of amino acid starvation by synthesizing GCN4, a typical eukaryotic transcriptional activator, which regulates the expression of many amino acids biosynthetic genes. By introducing point mutations in the DNA binding domain of GCN4, mutants with normal DNA binding activity but defective in transcriptional activity were isolated to identify unknown proteins that could suppress the mutant phenotype under an amino acid depletion condition. As a result, SSB(Stress-Seventy B) subfamily proteins were identified as suppressors of mutant GCN4. SSB proteins were known as a member of yeast hsp70 family that probably aids passage of nascent chain through ribosomes. Among them, the mechanism of suppression by SSB2 on the defective GCN4 mutant strains is under investigation. Gcn4p directly interacts with Ssb2p through the basic DNA binding domain of GCN4. It suggests the possibility that physical interaction might induce the transcriptional activation of Gcn4p.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.3 no.1
• /
• pp.15-18
• /
• 1998

The entanol productivity of superoxide dismutase (SOD)-deficient mutants of Saccharo-Myces cerevisiae was examined under the oxidative stress by Paraquat. It was observed that MnSOD-deficient mutant of S. cerevisiae had higher ethanol productivity than wild type or CuZnSOD-deficient yeast both in aerobic and in anaerobic culture condition. Pyruvated dehydrogenase activity decreased by 35% and alcohol dehydrogenase activity increased by 32% were observed in MnSOD-deficient yeast grown aerobically. When generating oxygen radicals by Paraquat, the ehanol productivity was increased by 40% in CuZnSOD-deficient or wild strain, resulting from increased activity of alcohol dehydrogenase and decreased a activity of pyruvate dehydrogenase. However, the addition of ascorbic acid with Paraquat returned the enzyme activities at the level of control. These results imply that SOD-deficiency in yeast strains may cause the metabolic flux to shift into anaerobic ethanol fermentation in order to avoid their oxidative damages by Paraquat.

• BMB Reports
• /
• v.34 no.2
• /
• pp.139-143
• /
• 2001

Thioredoxin (Trx) is a redox protein possessing conserved sequence Cys-Gly-Pro-Cys in ail organisms. Trx acts as an electron donor of many proteins including thioredoxin peroxidase (TPx). Yeast Trx 2 has two redox active cysteine residues at positions 31 and 34. To investigate the redox activity of each cysteine, we generated mutants C31S, C34S, and C31S/C34S using site directed mutagenesis and examined the redox activity of Trx variants as an electron donor for yeast TPx enzymes. None of the three Cysmutated Trx proteins was active as a redox protein in the 5', 5'-dithiobis-(2-dinitrobenzoic acid) reduction under the condition of the presence of NADPH and thioredoxin reductase, and in the thioredoxin dependent peroxidase activity of yeast TPx II. C34S enhanced the glutamine synthetase protection activity of yeast TPx I, even though 100 times more protein was needed to exhibit the same activity to WT. The formation of a mixed disulfide intermediate between Trx and TPx II subunits was analyzed by SDS-PAGE. The mixed dieter form of TPx II was found only for C34S. These results suggest that Cys-31 more effectively acts as an electron donor for TPx enzymes.

• Korean Journal of Microbiology
• /
• v.44 no.1
• /
• pp.80-84
• /
• 2008

We constructed the null mutants of fission yeast Schizosaccharomyces pombe spDbp5 gene that is homologous to DEAD-box RNA helicase DBP5 in budding yeast Saccharomyces cerevisiae, which plays important roles in mRNA export out of nucleus. A null mutant in an $h^+/h^+$ diploid strain was constructed by replacing the spDbp5-coding region with an $ura4^+$ gene using one-step gene disruption method. Tetrad analysis showed that the spDbp5 is essential for vegetative growth. The haploid spDbp5 null mutants harboring pREP81X-spDbp5 plasmid showed extensive $poly(A)^+$ RNA accumulation in the nucleus and decrease in the cytoplasm after repression of spDbp5 expression. These results suggest that spDbp5 is also involved in mRNA export from the nucleus.

• Korean Journal of Microbiology
• /
• v.44 no.2
• /
• pp.98-104
• /
• 2008

We constructed the null mutants of fission yeast Schizosaccharomyces pombe rsml gene that is thought to be involved in mRNA export. Though rsm1 gene is not essential for growth, the null mutant strain constructed by replacing the rsm1-coding region with an $kan^{r}$ gene showed growth retardation and mRNA export defects compared to wild type strain. We constructed double mutants which harbor rsm1 null allele and mutant allele of genes involved in mRNA export. The mex67 or npp106 null allele, when combined with rsm1 null allele, showed an additive effect on growth retardation and mRNA export defects. On the other hand, the thp1 null allele restored the defects of growth and mRNA export of rsm1 null mutant. These results suggest that rsm1 plays a role in mRNA export from the nucleus.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.9
• /
• pp.1573-1579
• /
• 2018

Transcriptional gene silencing is regulated by the chromatin structure, which is by various factors including histones. Saccharomyces cerevisiae contains transcriptionally silenced regions such as telomeric regions and hidden mating (HM) loci. The positively-charged amino acids on the histone H4 tail were reported to be critical for the telomeric silencing in yeast, by interacting with Dot1, a specific methyltransferase for the $79^{th}$ lysine on histone H3. However, Dot1 did not affect gene silencing within HM loci, but whether the positively-charged amino acids on the H4 tail affect HM silencing has not been defined. To elucidate the function of the H4 tail on HM silencing, we created several MATa-type yeast strains bearing the substitution of arginine with alanine or lysine on the histone H4 tail and checked the sensitivity of MATa-type yeast to alpha pheromone. The arginine point mutants substituted by alanine (R17A, R19A, and R23A) did not show sensitivity to alpha pheromone, but only two arginine mutants substituted by lysine (R17K and R19K) restored the sensitivity to alpha pheromone-like wild type. These data suggested that the basic property of arginine at $17^{th}$ and $19^{th}$ positions in the histone H4 tail is critical for maintaining HM silencing, but that of the $23^{rd}$ arginine is not. Our data implicated that the positive charge of two arginine residues on the histone H4 tail is required for HM silencing in a manner independent of Dot1.

• Journal of Microbiology
• /
• v.39 no.4
• /
• pp.286-292
• /
• 2001

During mitosis. the proper segregation of duplicated chromosomes is corrdinated by a spindle check-point. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the misorientation of the mitotic spin- dle Ibd1p/Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for mitotic exit and its disruption abolishes mitotic arrest when proper organization of the mitotic spin-dls inhibited. Ibd1p/Bfa1p localizes to the spindle pole body, a microtublue-organizing center in yeast, and its overexpression arrests the cell cycle in 80% of cells with an enlarged budy at mitosis and in 20 % of cells with multiple buds. In this study, we found that the C-terminus of Ibd1p/Bfa1p phys-ically interacts with Skp1p, a key component of SCF (Skp1/cullin/F-box) complex for ubiquition-medi-ated proteolysis of cel cycle regulatores as well as an evolutionally conserved kinetochore protein for cell cycle progression. A putative F-box motif was found in the C-terminus of Ibd1p/Bfa1p and its function was investigated by making mutants of conserved residues in the motif. These Ibd1p/Bfa1p mutants of a putative F-box interacted with SKp1p in vitro by two-hybrid assays as wild type Ibd1p/Bfa1p. Also these Ibd1p/Bfa1p utants displayed the overexpression phenotypes of wild type Ibd1p, when over-expressed under inducible promoters . These results suggest that a putative F-box motif of Ibd1p/Bfa1p is not essential for the interaction with SKp1p and its function in mitotic exit and cytokinesis.

• Microbiology and Biotechnology Letters
• /
• v.45 no.1
• /
• pp.81-86
• /
• 2017

Alignment of 967 reference sequences of the typical 2-Cys peroxiredoxin family of proteins revealed that 10 amino acids were conserved, with over 99% identity. To investigate whether the conserved aspartic acid residues and serine residue affect the peroxidase and chaperone activity of the protein, we prepared yeast TSA1 mutant proteins in which aspartic acids at positions 75 and 103 were replaced by valine or asparagine, and serine at position 73 was replaced by alanine. By non-reducing SDS-PAGE, TSA1 and the S73A, D75V and D75N mutants were detected in dimeric form, whereas the D103V and D103N mutants were detected in various forms, ranging from high molecular-weight to monomeric. Compared with wild type TSA1, the D75N mutant exhibited 50% thioredoxin peroxidase activity, and the S73A and D75V mutants showed 25% activity. However, the D103V and D103N mutants showed no peroxidase activity. All proteins, except for the D103V and D103N mutants, exhibited chaperone activity at $43^{\circ}C$. Our results suggest that the two conserved aspartic acid residues and serine residue of TSA1 play important roles in its thioredoxin peroxidase activity, and D103 plays a critical role in its chaperone activity.

• Journal of Microbiology
• /
• v.41 no.2
• /
• pp.121-128
• /
• 2003

The identification of genes involved in true hypha formation is important in the study of mechanisms underlying the morphogenetic switch in yeast. We isolated a gene responsible for the morphogenetic switch in Yarrowia lipolytica, which forms true hyphae in response to serum or N-acetylglucosamine. The isolated gene, encoding 847 amino acids, had sequence identities of 27% and 25% with the Bud6 (Aip3) proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. Disruption of this gene, designated YIBUD6, in haploid and diploid strains significantly reduced the ability of Y. lipolytica to switch from the yeast form to the hyphal form in hypha-inducing media. It was also found that YIBud6$\Delta$ mutants were rounder than the wild type when grown in the yeast form. These results indicate that the YIBud6 protein is necessary for hyphal growth and cell polarity in both haploid and diploid Y. lipolytica cells.