Chapter 3 Isolation and subfractionation of mitochondria from plants

Techniques for the isolation of plant mitochondria, without changing the morphological structure observed in vivo and maintaining their functional characteristics, require methods that avoid osmotic rupture of membranes and protect organelles from harmful products released from other cellular compartments. This chapter provides an overview of classic methods and some more detailed examples of recent protocols for specialized purposes. Mitochondria from plants share significant similarities with those from other eukaryotic organisms in terms of both their structure and function. Plant mitochondria provide energy in the form of ATP, provide a high flux of metabolic precursors in the form of tricarboxylic acids to the rest of the cell for nitrogen assimilation and biosynthesis of amino acids, and have roles in plant development, productivity, fertility and susceptibility to disease. Study of the composition, metabolism, transport processes, and biogenesis of plant mitochondria requires an array of procedures for the subfractionation of mitochondria to provide information on localization and association of proteins or enzymatic activities. This chapter also presents subfractionation procedures employed for the separation of mitochondria into four compartments. Further techniques for separation and partial purification of membrane protein complexes are outlined in the chapter. Two-dimensional profiling of the mitochondrial proteome and identification of proteins by the use of antibodies, tandem mass spectrometry (MS), and in organelle [35S]methionine translation assays are discussed with examples.