Research Paper Volume 5, Issue 7 pp 551—574

Figure 3. Mitochondria-associated LCA is confined mainly to the IMM, and also resides in the OMM. Cells were cultured in the nutrient-rich YP medium initially containing 0.2% glucosewith exogenously added 50 μM LCA in the presence of 1% DMSO (A and C) or in its absence (B and D). Purified mitochondria of cells that were taken at day 4 of cell culturing were subjected to fractionation using a swell-shrink procedure and subsequent equilibrium density gradient centrifugation (A and B) or to fractionation using sonication and subsequent differential centrifugation (C and D). (A and B) The percent recoveries of loaded protein and LCA in sucrose gradient fractions are presented. Equal volumes of gradient fractions were subjected to lipid extraction followed by mass spectrometric identification and quantitation of LCA in the extracts of lipids. Equal volumes of gradient fractions were also analyzed by immunoblotting with antibodies to Por1p (a protein marker of the OMM), Ccp1p (a protein marker of the IMS), Cox2p (a protein marker of the IMM) and Mge1p (a protein marker of the mitochondrial matrix). (C and D) The percent recoveries of protein and LCA in the pellet of SMP (consisting of vesicular particles surrounded by the IMM and OMM resealed in the inside-out orientation) and the supernatant (containing protein and other components of the mitochondrial matrix and IMS); the pellet and supernatant fractions were recovered after high-speed centrifugation of sonicated mitochondria. Data are presented as means ± SEM (n = 3; *p < 0.01).