Purification and Subfractionation of Mitochondria from the Yeast Saccharomyces cerevisiae

This chapter focuses on the well-established protocols for isolating yeast mitochondria, purifying the organelle using Nycodenz gradients, determining the integrity of isolated mitochondria, and fractionating mitochondria into their sub-compartments. It discusses several protocols for isolation of mitochondria such as growth of yeast cells, isolation of crude mitochondria, and purification of crude mitochondria using continuous Nycodenz gradient centrifugation. It describes how these methods may be used to determine whether a protein localizes to mitochondria and to sub-compartments (outer membrane, inner membrane, inter-membrane space, and matrix) within the organelle. Isolated mitochondria can be fractionated further into inner and outer membranes, as well as contact sites, which are sites of close contact between the outer and inner membranes that are implicated as sites for translocation of protein and phospholipids across, and between, the mitochondrial outer and inner membranes. Isolated mitochondria are routinely used to study activities of resident mitochondrial proteins and determine whether a protein localizes to mitochondria. For both applications, it is critical to document the purity and integrity of the mitochondrial preparation. The simplest way to do so is to perform SDS-PAGE and Western blot analysis, using antibodies raised against proteins of different membrane organelles. The chapter also describes the methods to determine the disposition of proteins on mitochondrial membranes.