Evaluation of genes involved in oxidative phosphorylation in yeast by developing a simple and rapid method to measure mitochondrial ATP synthetic activity

Microbial Cell Factories

Technical Notes

Akihiko Kondo¹  Keiji Nishida²  Xiaoting Ye²  Kana Morikawa²  Tomohisa Hasunuma²  Kiyotaka Y Hara²  Shih-Hsin Ho²  Michihiro Araki² 

[1] Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, 657-8501, Nada, Kobe, Japan;Organization of Advanced Science and Technology, Kobe University, 657-8501, Nada, Kobe, Japan;

关键词: Mitochondria purification;    ATP synthesis;    High-throughput analysis;   

DOI  :  10.1186/s12934-015-0239-z

 received in 2014-12-14, accepted in 2015-04-02,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundMeasurement 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.ResultsTo 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.ConclusionsCompared 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.

【 授权许可】

Unknown   
© Ye et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.