Research Paper Volume 5, Issue 7 pp 551—574

Figure 2. Intracellular LCA accumulates in mitochondria.

Cells were cultured in the nutrient-rich YP medium initially containing 0.2% glucose with exogenously added 50 μM LCA in the presence of 1% DMSO (A and B) or in its absence (C and D). Homogenates of cells that were taken at day 4 (A and C) or 7 (B and D) of cell culturing were subjected to subcellular fractionation to recover a mix of mitochondria, endoplasmic reticulum (ER), Golgi, vacuoles, plasma membrane (PM) and nuclei in a 12,000 × g pellet. The recovered mix of organelles was fractionated using centrifugation to equilibrium in a sucrose density gradient. 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 mitochondria), Nsp1p (a protein marker of the nucleus), Dpm1p (a protein marker of the ER), Vps10p (a protein marker of the Golgi), Pma1p (a protein marker of the PM) and Pho8p (a protein marker of the vacuole).