Ye Xiaoting, Morikawa Kana, Ho Shih-Hsin, Araki Michihiro, Nishida Keiji, Hasunuma Tomohisa, Hara Kiyotaka Y, Kondo Akihiko
Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe, 657-8501, Japan.
Microb Cell Fact. 2015 Apr 16;14:56. doi: 10.1186/s12934-015-0239-z.
Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. However, fractionation of mitochondria requires large amounts of cells, lengthy purification procedures, and an extreme caution to avoid damaging intact mitochondria, making it the highest barrier to high-throughput studies of mitochondrial function. To evaluate 45 genes involved in oxidative phosphorylation in Saccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis.
To obtain functional mitochondria, S. cerevisiae cells were lysed with zymolyase followed by two-step, low- then high-speed centrifugation. Using a firefly luciferin-luciferase assay, the ATP synthetic activity of the mitochondria was determined. Decreasing the ATP synthesis in the presence of mitochondrial inhibitors confirmed functionality of the isolated crude mitochondria. Deletion of genes encoding mitochondrial ATP synthesis-related protein showed their dependency on the oxidative phosphorylation in S. cerevisiae.
Compared with conventional procedures, this measurement method for S. cerevisiae Mitochondrial ATP Synthetic activity in High-throughput (MASH method) is simple and requires a small amount of cells, making it suitable for high-throughput analyses. To our knowledge, this is the first report on a rapid purification process for yeast mitochondria suitable for high-throughput screening.
测量线粒体ATP合成是比较细胞能量代谢性能的关键方法。然而，线粒体分级分离需要大量细胞、冗长的纯化程序，并且要极其小心以避免损坏完整的线粒体，这使其成为线粒体功能高通量研究的最大障碍。为了评估酿酒酵母中参与氧化磷酸化的45个基因，我们旨在开发一种简单快速的方法来测量线粒体ATP合成。
为了获得功能性线粒体，用溶菌酶裂解酿酒酵母细胞，然后进行两步离心，先低速后高速。使用萤火虫荧光素-荧光素酶测定法测定线粒体的ATP合成活性。在存在线粒体抑制剂的情况下ATP合成减少证实了分离的粗制线粒体的功能。编码线粒体ATP合成相关蛋白的基因缺失表明它们在酿酒酵母中对氧化磷酸化的依赖性。
与传统方法相比，这种用于酿酒酵母线粒体ATP合成活性高通量测量的方法（MASH方法）简单且需要少量细胞，使其适用于高通量分析。据我们所知，这是关于适用于高通量筛选的酵母线粒体快速纯化过程的首次报道。
