Regulation of Mitochondrial Biogenesis in Yeast

总结 我们的长期目标是了解线粒体的分子机制， 生物起源线粒体产生大部分细胞的三磷酸腺苷（ATP）， 作为真核细胞中的"能量货币"。除了它们的代谢功能，线粒体 参与各种各样的（病理）生理过程，例如细胞凋亡，癌症， 退行性疾病和衰老。Hap 2/3/4/5复合物是一种主要的转录激活因子 线粒体生物合成的关键。Hap4是该复合物的调节亚基， 水平决定复合物的活性。我们的初步数据表明， 酵母中的生物合成是通过转录和翻译后调节介导的， Hap4.我们发现Hap4的周转是通过泛素蛋白酶体系统介导的， 需要两种泛素结合酶，Ubc1和Ubc4。Ubc 4的半胱氨酸残基22是 在其直系同源物中高度保守，我们发现它是还原剂- Ubc4和Hap4周转的敏感修饰。我们还发现了七种不同的顺式作用 HAP4启动子中对HAP4表达重要的元件。在本提案中，我们 本论文的主要目的有两个：1）表征氧化改性， ubc4及其在实现细胞氧化还原平衡中的潜在作用。我们将纯化Ubc4， 通过质谱法测定其氧化修饰。Ubc4修饰的阐明将 阐明了细胞如何通过调节线粒体生物合成来实现氧化还原稳态。（二） 为了表征HAP 4启动子和反式作用调节因子如何共同作用， 实现最佳HAP4表达和线粒体稳态。结合使用 生物化学，遗传学和细胞生物学工具，我们将确定的机制， 线粒体生物发生受细胞能量需求，介体复合物， 以及线粒体的功能状态。深入了解细胞如何实现功能性稳态 可能导致更好的医疗干预来治疗线粒体疾病。

Summary Our long-term goal is to understand the molecular mechanism underlying mitochondrial biogenesis. Mitochondria produce the majority of cells’ adenosine triphosphate (ATP) to be used as “energy currency” in eukaryotic cells. Apart from their metabolic function, mitochondria participate in a diverse array of (patho)physiological processes such as apoptosis, cancer, degenerative diseases, and aging. The Hap2/3/4/5 complex is a master transcriptional activator of mitochondrial biogenesis in yeast. Hap4 is the regulatory subunit of this complex and its levels determine the activity of the complex. Our preliminary data indicate that mitochondrial biogenesis in yeast is mediated through transcriptional and post-translational regulation of Hap4. We found that Hap4 turnover is mediated through the ubiquitin proteasome system and requires two ubiquitin-conjugating enzymes, Ubc1 and Ubc4. The cysteine residue 22 of Ubc4 is highly conserved among its orthologs and we found that it is required for both a reducing agent- sensitive modification of Ubc4 and Hap4 turnover. We also identified seven separate cis-acting elements in the promoter of HAP4 that are important for HAP4 expression. In this proposal, we are going to achieve the following two aims: 1) To characterize the oxidative modification to Ubc4 and its potential role in achieving cellular redox balance. We are going to purify Ubc4 and determine its oxidative modification via Mass Spectrometry. Elucidation of Ubc4 modification will shed lights on how cells achieve redox homeostasis by regulating mitochondrial biogenesis. 2) To characterize how the HAP4 promoter and trans-acting regulatory factors work together to achieve optimal HAP4 expression and mitochondrial homeostasis. Using a combination of biochemical, genetic, and cell biological tools, we will determine the mechanisms by which mitochondrial biogenesis is regulated by the cellular energetic demand, the Mediator complex, and the functional state of mitochondria. Insights into how cells achieve functional homeostasis of mitochondria may lead to better medical interventions to treat mitochondrial diseases.