title: Evaluation of genes involved in oxidative phosphorylation in yeast by developing a simple and rapid method to measure mitochondrial ATP synthetic activity.
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 in Saccharomyces 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 in S. cerevisiae.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan. xiaoting_ye@icloud.com.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Ye
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan. k-morikawa._.1023026@people.kobe-u.ac.jp.
authors.forename: Kana
authors.initials: K
authors.lastname: Morikawa
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan. stephen6949@shark.kobe-u.ac.jp.
authors.forename: Shih-Hsin
authors.initials: SH
authors.lastname: Ho
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan. araki@port.kobe-u.ac.jp.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Araki
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan. keiji_nishida@people.kobe-u.ac.jp.
authors.forename: Keiji
authors.initials: K
authors.lastname: Nishida
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan. hasunuma@port.kobe-u.ac.jp.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hasunuma
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe, 657-8501, Japan. kiyotaka.hara@dolphin.kobe-u.ac.jp.
authors.forename: Kiyotaka Y
authors.initials: KY
authors.lastname: Hara
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe, 657-8501, Japan. akondo@kobe-u.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D010085:Oxidative Phosphorylation
chemical_list: D000255:Adenosine Triphosphate