Evaluation of genes involved in oxidative phosphorylation in yeast by developing a simple and rapid method to measure mitochondrial ATP synthetic activity. Issue 1 (December 2015)

• Record Type: Journal Article
• Authors: Ye, Xiaoting; Morikawa, Kana; Ho, Shih-Hsin; Araki, Michihiro; Nishida, Keiji; Hasunuma, Tomohisa; Hara, Kiyotaka; Kondo, Akihiko
• Is Part Of: Microbial cell factories. Volume 14:Issue 1(2015)
• Page Start: 1
• Page End: 7
• Publication Date: 2015-12
• Subjects: Mitochondria purification -- ATP synthesis -- High-throughput analysis; Microbial biotechnology -- Periodicals; Recombinant proteins -- Synthesis -- Periodicals; 660.62
• DOI: 10.1186/s12934-015-0239-z
• Languages: English
• ISSNs: 1475-2859

Abstract

Abstract Background 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 inSaccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis. Results 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 inS. cerevisiae . Conclusions Compared with conventional procedures, this measurement method forS. 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.