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Formic acid is a weak organic acid which is fatal to human and is poisonous to yeast cell too. We prove that formic can induce a fast formation of ROS in yeast S.cerevisiae[2].so we use formic acid as ROS burst inducer and compare their levels in wild-type and yca1 mutant cells. ROS formation in cells of both two types is obvious.

B. Deletion of yca1 gene or inhibition of Yca1p activity accelerates the ROS burst

In log phase cells of S.cerevisiae, the deletion of yca1gene results a stronger ROS burst [2]. The inhibition of Yca1p activity also improved the ROS positive rate (Fig. 2). The death rate of WT cells is lower too (Fig. 2). So it seems that Yca1p can affect the ROS burst process.

Fig. 2 The level of ROS induced by formic acid in wild type and yca1 mutant cells of BY4741 background after 20 μM DCFH-DA staining,

which shows the ROS levels in cytoplasm. Cells were treated with 40mM formic acid for 80min in room temperature. The concentration of zVAD-

fmk is 16μM. The PI fluorescence was detected by FL2 channel.

C. Knock out of yca1 gene makes the yeast cell losing mitochondrial potential faster in the process of aging.

Fig. 3 yca1 mutants have lower mitochondrial membrane potential than wild type cells (BY4741 background) after 2 days cultivation in 3 . The

cells were stained by 1μM RH123 to show the mitochondrial membrane potential.

We find that in log phase there is no difference of mitochondrial membrane potentials between the WT and yca1 cells (data not shown). But with the aging of cells, the mitochondrial membrane potential of yca1 mutant is obviously lower (Fig. 3), which may be a consequence of more ROS damage.

D. Fluorescence of mtGFP in WT cells decreases faster than in yca1Δ cells during the process of ROS burst.

Fig. 4 Effect of ROS burst on the fluorescence intensity of ATPase-GFP in wild type and yca1 cells (BY4741 background). Cells were treated with formic acid for 0 min and 30 min in room temperature. The GFP fluorescence was detected by FL1 channel and PI fluorescence was

detected by FL2. The ATP synthase, which is located in the inner membrane of mitochondria, was fused with green fluorescent protein in S.cerevisiae. And the alteration of GFP was analyzed during the process of ROS burst. GFP is known to be quiet stable in cells, but the result indicated that the fluorescence of GFP disappeared quickly in this experiment, suggesting that the ROS burst could damage the GFP structure directly or indirectly. Knock out of yeast caspase 1 gene makes the mtGFP fluorescence vanish faster in the process of ROS burst (Fig. 4).So it seems that yeast caspase 1 may involved in the damage of GFP.

IV. CONCLUSION & DISCUSSION

Without the activity of yeast caspase 1, the ROS in cells can reach a higher level and lead to more cell death during ROS burst; Knock out of yeast caspase 1 gene accelerates the loss of mitochondrial membrane potential in aged cells; Knock out of yeast caspase 1 gene makes the decrease of mtGFP fluorescence slower in the process of ROS burst.

Recently years, people begin to know the none death roles of some caspases, such as cell proliferation,differentiation and stress response [3, 6, 8, 9].The positive effect of yeast metacaspase(Yca1p) to yeast cell is still a puzzle. We hypothesize that the aspartic enzyme activity of yeast caspase 1 form a highly hydrophilic terminal on the surface of oxidized protein and leads to fast degradation of these proteins during ROS burst. And the hydrolyzed proteins can be more effective ROS scavenger. Besides, the caspase also take part in some other cellular changes to make the cell live through the vital stress of oxidization. This hypothesis may help to find out the meaning of yeast caspase

yca1 WT

M1 M1

38.8 29.5

yca1+zVAD

-fmk

0.7% 0.5%0.7%

76.9%22.0%

1.0%

89.6% 91.0%5.0%

2.6% 1.4%

0.5%

6.0%

95.5%

2.6% 1.8%

3.0%WT

yca1

WT +zVAD

- fmk

WT yca1

30min

0min

2010 International Conference on Bioinformatics and Biomedical Technology154

1 to the survival of yeast cell, and to explain some morphological characters of cell apoptosis. Yeast caspase 1, which may also have an aerobic and endosymbiotic bacteria evolutionary origin, probably belongs to a conserved antioxidation mechanism.

REFERENCES

[1] Boyce, M., Degterev, A., and Yuan, J. (2004). Caspases: an ancient cellular sword of Damocles. Cell Death Differ 11, 29-37.

[2] Du,Lin; Su,Yingying; Sun,Dongbei; Zhu,Wenhan; Wang,Jiayi; Zhuang, Xiaohong; Zhou, Shining; Lu, Yongjun. Formic acid induces Yca1p-independent apoptosis-like cell death in the yeast Saccharomyces cerevisiae, FEMS Yeast Research, 2008 4 , 531 – 539

[3] Galluzzi L, Joza N, Tasdemir E, Maiuri MC, Hengartner M, Abrams JM, Tavernarakis N, Penninger J, Madeo F, Kroemer G (2008) No death without life: vital functions of apoptotic effectors. Cell Death Differ 15:1113–1123.

[4] Khan, M. A., Chock, P. B., and Stadtman, E. R. (2005). Knockout of caspase-like gene, YCA1, abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 102, 17326-17331.

[5] Koonin, E. V., and Aravind, L. (2002). Origin and evolution of eukaryotic apoptosis: the bacterial connection. Cell Death Differ 9, 394-404.

[6] Lamkanfi M, Festjens N, Declercq W, Vanden BT, Vandenabeele P (2007) Caspases in cell survival, proliferation and differentiation. Cell Death Differ 14:44–55.

[7] Lee, R. E., Puente, L. G., Kaern, M., and Megeney, L. A. (2008). A non-death role of the yeast metacaspase: Yca1p alters cell cycle dynamics. PLoS ONE 3, e2956.

[8] Lim, H. W., Kim, S. J., Park, E. H., and Lim, C. J. (2007). Overexpression of a metacaspase gene stimulates cell growth and stress response in Schizosaccharomyces pombe. Can J Microbiol 53, 1016-1023.

[9] Yi CH, Yuan J (2009) The jekyll and hyde functions of caspases. Dev Cell,16:21–34.

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