ISOLATION OF GIANT MITOCHONDRIAL NUCLEOIDS FROM THE YEAST SACCHAROMYCES-CEREVISIAE

The yeast cells Saccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presen ce of a respiratory inhibitor, antimycin A, had distinctive giant mito chondrial nucleoids (mt-nucleoids) (apparent diameter 0.6-0.9 mu m) in contrast with the small mt-nucleoids (apparent diameter 0.2-0.4 mu m) in respiratory-sufficient cells grown aerobically, as revealed by DAP I-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 irregula r size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully iso lated from spheroplasts of three different cells by differential centr ifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were fre e of significant contamination by nuclear chromatin. The number of pro tein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grow n cells, though a few noticeable differences were also recognized. DNA -binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids 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 mt-nucleoids from anaero bically grown cells and from rho-cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of an timycin A seemed to lack the 67 kDa protein bur to have a small amount of the 52 kDa protein. This is the first demonstration of the varianc e of protein species involved in yeast mt-nucleoids according to the r espiratory activity of mitochondria.