Wild type strain (WT) and YDL100c disrupted strain (KO) were grown at 30oC for 8 hr after adding 50 £gM menadione. Cells of both strains were assayed for trehalose accumulation, intracellular molecular oxidation level, membrane lipid oxidation, and glutathione (GSH) content. The data showed both the molecular and membrane lipid oxidation levels are higher and the GSH content is lower in KO compared with WT in the presence of menadione. The catalase activity in KO strain are reduced than that in WT strain, catalase activity would affect in the presence of menadione. Further study of antioxidant gene expressions showed that TPS1 and CTT1 were involved in the general stress response¡FSOD1, GSH1, TRR1 and TRX2 were involved in the specific stress response. Above mentioned mRNA level were reduced, suggesting that the deletion of YDL100c in S. cerevisiae affects the operation of general and specific stress response when grown in the presence of menadione.

Abstract ArsA protein is the catalytic component of the bacteria plasmid R773-encoded ArsAB pump that is in involved in As3+ detoxification. Homologues of the ArsA protein are found in nearly all organisms but the biological functions of these homolog proteins are still unclear. The ArsA homologue in S. cerevisiae is encoded by the ORF YDL100c. Initial studies show that deletion of YDL100c in S. cerevisiae was not lethal and had no effect on As3+ sensitivity at 30¢J. However, the disrupted strain (KO strain) is unable to grow at 40¢J and shows increased sensitivity to Co2+,Zn2+,As3+ and Sb3+ at 37¢J by spotting assay. In this study, a plasmid (YEp352) carrying the YDL100c under the control of its endogenous promoter was used to study the induction of YDL100c under various stress conditions. The data show that the expression of Ydl100cp increased 30 % at 37¢J compared to that at 30¢J, and the expression can be induced by low dosage of Zn2+, Ni2+, Sb3+ and neutral to alkaline pH. Overall, temperature is the best inducer for Ydl100cp expression. Besides, searching Ydl100cp in Internet yeast two hybrid database and YDL100c promoter sequence analysis database suggest the following experiments and results:¡]1¡^2D gel electrophoresis assay to demonstrate different protein patterns between WT and KO strain under nonpermissive temperature. ¡]2¡^Flow cytometry data indicate most of KO strain cells growth arrest at G2/M phase in nonpermissive temperature. ¡]3¡^Microscopic data reveal KO stain cells grew very densely and showed cluster phenotype at nonpermissive temperature. When Congo red was used to stain cell wall¡Ait was found that these cluster cells is actually one cell. Although the cell wall between mother and daughter cell can form cleavage furrow, the formation is not complete and cell can¡¦t separate into two individuals. Consequently, the cells grow densely with cluster form and mega-polynuclear cells. It suggests Ydl100cp is induced and plays a role in cell cycle under nonpermissive temperature. The function of Ydl100cp may be a late mitosis cyclin-like protein or cyclin dependent kinase inhibitor that controls several downstream genes related to cell wall formation, maintenance, and structure. Because KO strain does not have Ydl100cp, it shows different growth patterns compared to WT strain when grow at nonpermissive temperature. Initial studies suggest that YDL100c is involved in general responses because KO strain shows sensitivity to a broad range of metals. However, based on the results have, it is possible that Ydl100cp is involved in cell wall structure, formation and maintenance. Under nonpermission temperature cell wall of KO strain had defect that led to defect in ion transport structure. Therefore cell can remove not only can not poison metals especially Zn2+, Ni2+, Co2+, arsenite and antimonite metals right away but these metals can also pass cell wall into cytoplasm that causes KO strain reveals sensitivity to metals. To sum up the results, the expression of Ydl100cp can be induced under nonpremssive temperature to decrease mega-polynuclear cells formation and control downstream genes for cell wall formation, maintenance and structure. Therefore yeast cells can survive at nonpermissive temperature instead to be killed.

ArsA is the catalytic component of an arsenite extrusion pump in E. coli that confers arsenite and antimonite resistance. YDL100cp is the ArsA homologous protein found in S. cerevisiae. Previous studies show that YDL100c gene is not directly related to arsenical resistance mechanism in S. cerevisiae but the YDL100c disrupted strain (KO) showed sensitivity to Zn2+ at 30oC and more pronounced sensitivity at 37oC. To study the role of YDL100c on Zn2+ sensitivity, wild type strain (WT) and KO were grown at 30oC and 37oC for 6 hr after adding Zn2+. Both strains were assayed for trehalose accumulation, intracellular oxidation level and GSH content. The results demonstrate that KO had a decreased growth and increased intracellular oxidation at 37oC when compared to WT. Addition of Zn2+ did not increase the intracellular oxidation in WT and KO grown at 30oC but to a greater extent in KO compared to WT grown at 37oC. Further assess the function of antioxidant genes shows that there is no significant difference in SOD1 expression between KO and WT grown at 37oC but CTT1 expression is low in KO. There is an increase in catalase activity for both WT and KO by adding Zn2+ at 30oC or 37oC, but the level of catalase activity to KO is still lower than that of WT.In conclusion, a defect of YDL100c results in a defect in the activation of general stress response at 37oC. Consequently, the cause of the increased level of intracellular oxidation of KO in the presence of Zn2+ grown at 37oC is most likely related to the decrease in cellular GSH content and trehalose accumulation in KO compared to that of WT. Therefore, the pronounced sensitivity to Zn2+ at 37oC is mainly due to a defect in general stress response in KO when grown at 37oC.

YDL100Cp is the ArsA homologue protein found in S. cerevisiae. In bacteria, ArsA protein is involved in As3+detoxification but the function of YDL100Cp is still unknown. Previous studies show that deletion of YDL100C in S. cerevisiae was not lethal and had no effect on As3+ sensitivity or growth at 30¢J. However, when grown at 40¢J, growth of YDL100C disrupted strain (JSY1) was inhibited. To study the role of YDL100C in response to lethal heat shock, wild type (W303-1B) and JSY1 cells were exposed to 50¢J for 15 min. The survival rate of JSY1 cells was half of W303-1B cells and the difference in survival rate was complemented by introduction of plasmid carrying YDL100C. It suggests that YDL100Cp plays a role in acquisition of thermotolerance to lethal heat shock. It is believed that there are two factors involved in heat-induced cell death: the heat damage and the oxidative damage. Determinations of heat-damage related defense system in S. cerevisiae, including trehalose (a thermoprotectant) content, Hsp70 expression and Hsp104 expression, demonstrate that heat damage should not be the major cause of JSY1 cell death during heat shock. For the oxidative damage, the measurement of in vivo reactive oxygen species reveal the lower protein damage caused by reactive oxygen species (ROS) in JSY-1 after 50¢J 15 min heat shock, this might reflect the difference in viability of three strains under lethal heat shock. And with the intra cellular content of glutathione, it revels that the YDL100C deficient caused cell got more serious oxidative damage under 50¢J heat shock. But the observation of thermotolerance related ROS scavenger system (including the catalase, and superoxide dismutase) expression with reverse transcription polymerase chain reaction suggested that YDL100C deficient had no effect on triggering these system. As the result, it is suggested that the function of YDL100Cp in S. cerevisiae might be an oxidative damage repair system, such as the glutathione peroxidase. It might react with the oxidative damage substance and function as a deoxidizer.

YDL100cp is the ArsA homologous protein found in Saccharomyces cerevisiae. Previous studies show that deletion of YDL100c was not lethal but unable to grow at 40¢XC. To study the role of YDL100c in response to lethal heat shock, the wild type strain (WT) and YDL100c disrupted strain (KO) were exposed to 50¢XC for 30 min. The growth and survival rate of KO cells at 30¢XC after heat-shock was lower than that of WT cells, and the difference was complementated by introducing the plasmid carrying YDL100c. The oxidative stress has been shown to be involved in the heat-induced cell death in S. cerevisiae. Therefore, the intracellular molecular oxidation level, expression of antioxidant genes, trehalose accumulation, and glutathione (GSH) content were further examined. The intracellular molecular oxidation was increased in KO compared to WT when exposed to 50¢XC, suggesting heat-shock-induced cell death is related to oxidation of intracellular components. The results also demonstrated that both WT and KO had a decreased GSH content and trehalose accumulation after heat-shock, indicating that GSH and trehalose are not directly involved in the slow growth of KO after heat-shock. However, CTT1 expression is decreased in KO compared to WT when exposed to 50¢XC, suggesting that decreased CTT1 expression resulted in the increased intracellular oxidation and YDL100c is likely involved in the activation of CTT1 expression.

To study the role of YDL100c during the growth of Saccharomyces cerevisiae in the presence of oxidant, the wild type strain (WT) and YDL100c disrupted strain (KO) were grown at 30oC for 6 hr after adding 0.25 mM of tert-butyl hydroperoxide (t-BOOH). The cells of both strains were assayed for the expression of anti-oxidant system, trehalose accumulation, intracellular molecular oxidation level, membrane lipid peroxidation, and glutathione (GSH) content. The results show that growth of KO is slower than that of WT and the cause of growth delay is the cell death. The data also show that the molecular oxidation level is lower but the lipid peroxidation of membrane is higher in KO compared with WT in the presence of t-BOOH, indicating that ROS do cause the damage on membrane. Further, analysis of the expression of cellular defense-related genes show that expressions of GSH1, CTT1, TPS1, TSL1, and NTH1 in KO are lower than in WT, but expressions of SOD1, TRR1 and TRX1 have no difference, demonstrating that the deletion of YDL100c in S. cerevisiae affects the general and specific stress response when grown in the presence of t-BOOH. In general, the decrease in CTT1 expression is not consistent with the catalase activity assay, however, decreased expressions of GSH1 and genes involved in trehalose metabolism are consistent with the decreased GSH content and increased trehalose accumulation in KO compared with WT. Therefore, the cause of KO cell death in the presence of t-BOOH is most likely related to the decrease in cellular GSH level and trehalose accumulation.