26] Yeast mitochondria and submitochondrial particles

Abstract

Mitochondria and submitochondrial particles have been prepared from several species of yeast, including Candida (Torulopsis) utilis, Saccharomyces cerevisiae, and closely related species such as S. carlsbergensis. The foremost experimental obstacle in the preparation of intact yeast mitochondria is the need for efficiently breaking the refractory cell wall without extensively damaging the liberated mitochondria. Two approaches to this problem have been used: (1) Cells are ruptured mechanically and mitochondrial fragments are separated from more intact mitochondria by differential centrifugation and careful separation of supernatants and loosely packed (fluffy) layers (submitochondrial particles) from firmer pellets (mitochondria). (2) Yeast cells are subjected to enzymatic cell wall digestion in order to form osmotically sensitive spheroplasts (protoplasts), from which mitochondria are liberated by osmotic shock and mild homogenization. Several mechanical devices have been employed for breaking yeast, including various shakers, the French pressure cell, blendors with overhead drive, and certain types of mills. All these devices under appropriate conditions have yielded yeast mitochondria or submitochondrial particles capable of oxidative phosphorylation, but in only a few instances has acceptor control of respiration been demonstrated with such preparations. The procedures described in the chapter employ a colloid mill; one method yields mitochondria with acceptor control.