Isolation of giant mitochondrial nucleoids from the yeastSaccharomyces cerevisiae

Abstract

Summary. The yeast cells Saccharomyces cerevisiae grown up tostationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6-0.9 ~tm) in contrast with the small mt-nucleoids (apparent diameter 0.2-0.4 ~m) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho-cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mtnucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNAbinding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mtnucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mtnucleoids from anaerobically grown cells and from rho-cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.

Dynamic Changes in Mitochondrial Nucleoids during the Transition from Anaerobic to Aerobic Culture in the Yeast Saccharomyces cerevisiae

“…Anaerobic culture was performed as previously described (Shiiba et al 1997). The AN medium used was basically the same as that in Tustanoff and Bartley (1964).…”

“…To isolate mt-nucleoids from cells during the transition from anaerobic to aerobic culture, the spheroplasts were first disrupted with a Teflon Potter-Elvehjem homogenizer instead of a Waring blender in order to minimize contamination by nuclear chromatin, after which the mt-nucleoids were isolated by the conventional method (Shiiba et al 1997). To compare mt-nucleoid proteins between aerobic and anaerobic conditions, cells were grown aerobically to the stationary phase in modified Burkholder's medium (BT medium) and mt-nucleoids were isolated by a method described previously (Miyakawa et al 1987).…”

“…The pellet of mt-nucleoids was suspended in 400 ml of TAN buffer (0.5 M sucrose, 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 7 mM 2-mercaptoethanol, 0.4 mM PMSF) and treated as follows (Shiiba et al 1997). The suspension was mixed with 400 ml of TAN buffer containing 4 M NaCl, to which were added 200 U of DNase I (type DSV; Worthington Corp., Freehold, NJ, USA), 10 mM MgCl 2 (final concentration), and 0.4 mM PMSF (final concentration).…”

“…Spheroplasts of anaerobically grown cells were formed and giant mt-nucleoids were isolated from them as previously described (Shiiba et al 1997).…”

“…For this reason, this yeast has long been used to study the correlation between the fine structure of mitochondria and their respiration activity (Stevens 1981, Pon andSchatz 1991). So far, using DAPI-fluorescence microscopy, we have revealed the dynamic morphological changes that mt-nucleoids undergo during S. cerevisiae's life cycle (vegetative growth, sporulation, mating, and germination) (Sando et al 1981, Miyakawa et al 1984, 1994 and have found that giant mt-nucleoids are formed in respiratory-deficient rho Ϫ cells (Miyakawa et al 1988) and in anaerobically grown cells (Shiiba et al 1997). We have developed methods for isolating giant mt-nucleoids from yeast cells cultured under anaerobic conditions, and have reported that quantities of two DNA-binding proteins, one 67 kDa and the other 52 kDa, among other mt-nucleoid proteins, were greatly lower in giant mt-nucleoids than in small mt-nucleoids from aerobically grown cells (Shiiba et al, 1997).…”