Isolation and subfractionation of mitochondria from plants

Publisher Summary This chapter presents various techniques used for the isolation of plant mitochondria, which maintains their morphological structure observed in vivo and their functional characteristics. These techniques outline a basis for studying plant mitochondria at the level of overall metabolic function, individual enzyme and protein complex composition and operation, and each polypeptide chain in the organelle proteome. Using a combination of osmotic shock and differential centrifugation, plant mitochondrial samples can be fractionated into four components. These comprise the two aqueous compartments (the matrix and the intermembrane space)— and the two membrane compartments(the inner mitochondrial membrane (IM) and the outer mitochondrial membrane. To perform a sub-fractionation, a purified mitochondrial sample, which has not been frozen, is separated into two aliquots, and each is added to 49 times the volume of two separate solutions. Samples of IM can be separated by two simple chromatography columns to provide relatively high purity fractions containing functional components of the electron transport chain and the mitochondrial superfamily of transporters. The chapter also describes the profiling of the mitochondrial proteome and identification of proteins by the use of mass spectrometry, antibodies, and in organello [ 35 S] methionine translation assays, along with examples.