探討FMN1和SET1在酵母菌粒線體中扮演的角色

The role of Riboflavin kinase and SET1 mediated H3K4 methylation in yeast mitochondria.

劉子瑜(Tzu-Yu Liu)

指導教授 ： 羅翊禎

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2017年)

https://doi.org/10.6342/NTU201700588

摘要

粒線體對於許多物種之存活扮演重要的角色，其功能包括參與ATP合成、鈣離子緩衝以及調節細胞凋亡等。維生素B2 (核黃素；Riboflavin) 會藉由FMN1轉錄之Riboflavin kinase合成黃素單核苷酸 (Flavin mononucleotide；FMN)，FMN會藉由FAD synthetase合成黃素腺嘌呤雙核苷酸 (Flavin adenine dinucleotide；FAD)，上述路徑發生在粒線體，且FAD為構成酵母菌粒線體complex I和II之輔酶，因此FMN1可維持酵母菌粒線體功能完整性。SET1會負責組蛋白H3上面第4個胺基酸Lysine (H3K4) 之甲基化作用，調節基因表現量。目前尚未有研究指出SET1在粒線體中的功能，但我們先前研究發現，fmn1-105, set1∆變異株粒線體功能相關之基因表現會被向下調控，例如ATP5 (ATP合成基因)、COQ2 (輔酶Q合成基因) 等。本研究目的為探討FMN1和SET1在粒線體中扮演之角色。研究發現， fmn1-105, set1∆突變株在高溫下存活率與利用不可發酵碳來源之能力皆顯著低於正常細胞 (WT)，但添加5 mM FMN可回復其功能。同時，fmn1-105, set1∆突變株粒線體DNA copy number顯著高於WT，且剔除TPK3 (酵母菌粒線體增生因子) 會減少fmn1-105, set1∆突變株粒線體DNA copy number增生，且降低不可發酵碳來源之利用以及對抗過氧化氫 (H2O2) 能力。另外，為了研究粒線體功能完整性對於外來氧化壓力之抵抗性，本研究亦使用七種藥物探討對於菌株對抗H2O2之影響，發現Rotenone (粒線體complex I 抑制劑) 與Antimycin A (粒線體complex III抑制劑) 會使fmn1-105, set1∆突變株對於H2O2之敏感性增高。另外也發現，以Rotenone處理後之fmn1-105, set1∆tpk3∆突變株對抗H2O2之能力顯著高於WT。推測Rotenone與Antimycin A會增加酵母菌內生性ROS。而TPK3基因剔除會使ROS上升或是下降，至今尚無定論。因此，Rotenone是否為藉由提高fmn1-105, set1∆tpk3∆突變株之ROS含量以協助其對抗氧化壓力，仍尚待研究。

關鍵字

粒線體功能失調 ； FMN1 ； SET1 ； TPK3 ； Rotenone ； Antimycin A ； H2O2

並列摘要

Mitochondria play important roles to all organisms, including ATP synthesis, calcium buffering, and apoptosis. FMN1 in Saccharomyces cerevisiae encodes riboflavin kinase which converts vitamin B2 (riboflavin) to flavin mononucleotide (FMN). FMN can be further converted to flavin adenine dinucleotide (FAD) by FAD synthetase. The metabolic pathway is occurred in yeast mitochondria, and FAD is the coenzyme of yeast mitochondrial complex I and II. So FMN1 is essential for maintenance of yeast mitochondria. SET1 is responsible for the lysine 4 methylation of histone H3 (H3K4), which play a role in gene transcription. We previously identified a temperature-sensitive FMN1 allele: fmn1-105, which renders synthetic lethality with set1∆. Our study showed that FMN1 and SET1 could coordinate mitochondrial function of yeast. TPK3 regulates yeast mitochondrial biogenesis, we found that it was essential for fmn1-105, set1∆ to increase its mitochondrial DNA copy number, use of non fermentable carbon sources, and resistant to oxidative stress. However, mitochondrial complex I inhibitor, Rotenone, helps fmn1-105, set1∆tpk3∆ fight against H2O2 stress. Whether the phenomenon is ROS-mediated, it needs further investigation.

並列關鍵字

mitochondrial dysfunction ； FMN1 ； SET1 ； TPK3 ； hydrogen peroxide (H2O2) ； Rotenone ； Antimycin A

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