Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (method)

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces ce revisiae were prepared by osmotic swelling and mechanical disruption of mit ochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) w ith respect to the mitochondria, The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with e ndoplasmic reticulum was around 5% (protein-based). The very low phosphatid ylserine synthase activity present in the OMV preparation indicated that co ntamination with mitochondria-associated membranes (MAM) was negligible. Th e resistance of the outer membrane protein Tom40 to digestion by trypsin de monstrated the sealed nature and right-side out orientation of the OMV. Ana lysis of the phospholipid composition revealed that the contents of phospha tidylcholine and phosphatidylinositol are higher and the content of phospha tidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.