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

Xiaoting Ye,Akihiko Kondo,Tomohisa Hasunuma,Shih-Hsin Ho,Michihiro Araki,Keiji Nishida,Kana Morikawa,Kiyotaka Y Hara

doi:10.1186/s12934-015-0239-z

Abstract

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.

Highlights

Measurement of mitochondrial Adenosine 5′-triphosphate (ATP) synthesis is a critical way to compare cellular energetic performance
We evaluated 45 genes involved in oxidative phosphorylation for mitochondrial ATP synthesis in S. cerevisiae
Preparation of crude mitochondria by the MASH method In the conventional method of mitochondrial purification, yeast cells are subjected to mechanical homogenization or detergent treatment followed by differential centrifugation because the variable density of the organelles will allow separation of the mitochondria from the remaining cellular structures

Summary

Introduction

Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. 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. Mitochondria are central organelles controlling the life and death of the cell They participate in key metabolic reactions, synthesize the majority of ATP in a cell, and regulate a number of signaling cascades, including apoptosis [1]. Owing to the ease of genetic manipulation and its importance for bio-industry, the budding yeast Saccharomyces cerevisiae is an ideal organism for the study of many basic cellular mechanisms in eukaryotic cells. Their organelles can be rapidly enriched in sufficient quantities for the analysis of specific functions such as metabolite or protein transport. One of the biggest problems is that the fractionation of mitochondria requires large amounts of cells, long

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Results