Mitochondria: Methods for Preparation

Abstract

Mitochondria are the main energy providers of the cell. Procedures developed for the isolation of pure and intact mitochondria have contributed considerably to the understanding of the complex biochemical functions of mitochondria, as they permit the application of detailed biochemical and biophysical techniques to the investigation of this organelle. This article describes the principles underlying the procedures for the isolation of mitochondria from a large variety of tissues and the techniques for assessing the quality of mitochondrial preparations. Special emphasis is given to cell fractionation protocols for the isolation of mitochondria from model organisms, including Saccharomyces cerevisiae, Arabidopsis thaliana, cultured mammalian cells, and for the isolation of hydrogenosomes and mitosomes from amitochondriates. The special requirements for the determination of the mitochondrial proteome by high‐throughput mass spectrometry are discussed.

Key Concepts:

  • Cell fractionation is a summary term for the isolation of cell organelles and subcellular fractions from tissues and cultivated cells.

  • Equilibrium centrifugation, a centrifugation technique by which particles are separated by virtue of their different densities in density gradients, is the most common technique for the preparation of highly enriched mitochondrial fractions.

  • Differential centrifugation, a centrifugation technique by which subcellular particles are separated by virtue of their differences in sedimentation rates, is the most common technique of cell fractionation protocols.

  • The mitochondrial proteome represents the whole set of proteins found within mitochondria.

  • Mitosomes and hydrogenosomes are mitochondria‐related organelles from protists.

  • A model organism is an experimental organism that is representative for a whole group of species. Models that are easy to manipulate tend to be the most widely used.

  • A subcellular marker is a selected biomolecule that is confined to a specific organelle or suborganellar compartment and that may be used to determine the presence of this compartment in a subcellular fraction.

  • The subfractionation of mitochondria involves techniques that allow the physical separation of individual mitochondrial compartments.

  • The quality of a mitochondrial preparation is determined by the degree of other contaminating organelles, the level of specific mitochondrial activities and the degree of mitochondrial integrity.

Keywords: cell organelles; hydrogenosomes; mitochondria; mitosomes; model organism; oxidative phosphorylation; preparative centrifugation; proteomics; subcellular fractionation

Figure 1.

The structure of mitochondria. (a) Electron micrograph of a thin section of a muscle cell showing mitochondria. (Photograph kindly provided by Dr M. Schrader; magnification: x56 000.) (b) Schematic representation of a typical mitochondrion including a few representative examples of common proteins and enzymatic activities associated with a particular mitochondrial subcompartment. Abbreviations: AAA, ATPases associated with various cellular activities; ATP, adenosine triphosphate; DNA, deoxyribonucleic acid; mt, mitochondrial; TIM, translocator of the inner membrane; TOM, translocator of the outer membrane.

Figure 2.

Generalised flow chart for the preparation of intact mitochondria. Detailed step‐by‐step laboratory recipes for the preparation of mitochondria from individual tissues. Experimental details were taken from the standard protocol for the isolation of yeast mitochondria. For explanations see text. Abbreviation: ER, endoplasmic reticulum.

Figure 3.

Generalised flow chart for the preparation of submitochondrial fractions. Two different experimental approaches are depicted: (left side) gentle disruption of the mitochondrial outer membrane by swelling or digitonin treatment; (right side) complete lysis of mitochondria by sonication. For explanations see text.

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Further Reading

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Mühlenhoff, Ulrich(Sep 2010) Mitochondria: Methods for Preparation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002601.pub2]