Isolation of highly purified mitochondria from Saccharomyces cerevisiae

This chapter focuses on the isolation of highly purified mitochondria from Saccharomyces cerevisiae and the purification of mitochondria from a 10-liter culture of wild-type yeast cells, grown in a stirred-tank fermenter. A typical yield is 50 to 100 mg mitochondrial protein. The procedure can be scaled up or down as appropriate. For example, 20 to 25 mg mitochondrial protein is obtained from four l-liter cultures grown in Erlenmeyer flasks with vigorous aeration. Isolated mitochondria can be fixed and then processed for thin-section electron microscopy. Fixation can be achieved simply by adding glutaraldehyde from a concentrated stock solution. However, two modifications of this method facilitate the subsequent analysis. First, it is often desirable to shrink the inner membrane–matrix compartment by placing the mitochondria in a hypertonic solution. This approach gives an indication of the intactness of the organelles, because mitochondria will shrink only if the inner membrane is osmotically tight. In shrunken mitochondria, the contact sites between the two membranes are more easily visualized. Second, the addition of tannic acid during the fixation greatly enhances the contrast, particularly with membrane structures. © 1995 Elsevier Inc.