Purification of Mitochondria from Yeast Cells

Summary

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Abstract

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells¹. These complex organelles also play essential roles in apoptotic cell death², cell survival³, mammalian development⁴, neuronal development and function⁴, intracellular signalling⁵, and longevity regulation⁶. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

Protocol

Materials and methods

Yeast strains and growth conditions

The wild-type strain BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) was grown in rich YEPD medium (1% yeast extract, 2% peptone, 2% glucose). Cells were cultured at 30°C with rotational shaking at 200 rpm in Erlenmeyer flasks at a "flask volume/medium volume" ratio of 5:1.

Isolation of crude mitochondrial fraction

1. Grow 1 L of the wild-type strain BY4742 culture for 48 h.
2. Pour culture in 500-ml Nalgene centrifuge tubes. Balance the load and harvest cells by centrifugation for 5....

Discussion

This method enables the high-yield isolation of pure mitochondria from yeast cells. Mitochondria purified by this method are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. The described method yields mitochondria that are suitable for cell-free reconstitution of essential mitochondrial processes. These highly pure mitochondria can also be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome.......