Mitochondrion of yeast: ultrastructural evidence for one giant, branched organelle per cell.

Three-dimensional models constructed from 80 to 150 consecutive serial sections of entire yeast cells showed that all the separate mitochondrial profiles were cross sections through a single, branching, tubular structure about 50 to 60 micrometers in length and 200 to 600 nanometers in diameter. The data are contrary to conventional notions of mitochondrial size, form, and number per cell and should lead to a reassessment of mitochondrial genetics and biogenesis.

Cytochemical localization of catalase activity in yeast peroxisomes.

Diaminobenzidine oxidation product occurred in peroxisomes and in the intracristate spaces of mitochondria. The reaction was inhibited only in peroxisomes when 3-amino-1,2,4-triazole was present, but cyanide and azide inhibited deposition in both kinds of organelles.

Size-separation of yeast mitochondria in the zonal centrifuge.

Mitochondria, released from yeast spheroplasts and subjected to rate separation through sorbitol gradients in the zonal centrifuge, migrated in a wide symmetrical zone. Electron micrographs showed that the mitochondria had been resolved within the zone according to size. The mean mitochondrial diameter at the leading edge was approximately twice that at the trailing edge of the particle zone. Activities of the enzymes cytochrome oxidase, malate dehydrogenase, and reduced nicotinamide adenine dinucleotide- and d-lactate cytochrome c reductases were essentially uniform throughout the mitochondrial zone. Mitochondria from a vegetative-petite mutant had almost the same size distribution as the isogenic wild type, but with somewhat larger mean diameter and either absent or markedly reduced enzyme activities. Mixtures of wild-type and petite mitochondria produced sedimentation profiles showing overlap of particle populations with respect to mean sedimentation rates and mitochondrial diameters, as well as intermediate levels of enzyme activities. Both cristate and noncristate organelles were present throughout the mitochondrial zone from these mixtures. Mitochondria centrifuged in sorbitol density gradients were well-preserved and yielded consistent sedimentation profiles, whereas particles in sucrose density gradients migrated more slowly, produced varied sedimentation profiles, and often showed spurious peaks, presumably due to particle aggregations.

Nuclear and mitochondrial DNA from wild-type and petite yeast: circularity, length, and buoyant density.

Purified mitochondrial and nuclear DNA from diploid isogenic wild-type and vegetative-petite baker's yeast were analyzed by electron microscopy and by analytical ultracentrifugation in CsCl gradients. The buoyant densities in CsCl of nuclear DNA were identical for the two strains (rho = 1.700), but there was a difference between mitochondrial DNA from the wild type (rho = 1.684) and the petite (rho = 1.680). Electron microscopy revealed both circular and linear filaments for nuclear and for mitochondrial DNA of both strains. Nuclear DNA molecules included 6.5 per cent cyclic filaments principally measuring 2 mu or less in contour length, and linear filaments showing a unimodal, disperse length-distribution centered at about 2 to 3 mu, for both strains. Mitochondrial DNA for wild type varied depending upon the method used to extract and purify the molecules; showing only 7.5 per cent circular molecules from CsCl-subfractionated samples, as compared with 15 per cent circles from chloroform-extracted DNA not subjected to CsCl and up to 50 per cent circles from osmotically-lysed mitochondira, as reported in an earlier study. Modal lengths of circles occurred at about 2, 5, and 10 mu Increasing shear degradation also was evident in comparisons of the length-distribution patterns of linear molecules using the three preparative methods. Petite mitochondrial DNA contained 36-38 per cent circular molecules which measured 0.3-5.3 mu, but principally in the range of 0.3 to 2.0 mu whether from chloroform-extracted populations or from ones subfractionated in CsCl. A previous study of osmotically lysed mitochondria had shown a maximum of 8 per cent circles, which we now attribute to a failure, at that time, to detect circles measuring less than 1 mu, a substantial component encountered in the purified DNA samples in the present study. Linear filaments presented a unimodal length distribution in every case. Despite the variation in molecule populations derived from the three different preparative methods, there were consistent differences between mitochondrial DNA from wild-type and petite yeast in frequencies and size of circular molecules, as well as in length distribution patterns.

Distribution of cytochrome c peroxidase activity in wild-type and petite cells of bakers' yeast grown aerobically and anaerobically.

Studies of mitochondrial biogenesis in yeast have been hampered by a lack of suitable membrane markers in anaerobically grown cells subsequently grown in air. Cytochrome c peroxidase activity and subcellular location was studied to determine whether it would be a useful marker for an analysis of mitochondrial formation. Cytochemical tests revealed enzyme reaction product on all mitochondrial membranes in aerobically grown wild-type cells. Anaerobically grown wild-type and all petite cultures contained cytochrome c peroxidase cytochemical reaction deposits on abundant cytoplasmic membranes and on the few mitochondrial profiles which also were seen in the electron photomicrographs. Biochemical studies corroborated the cytochemistry because mitochondrial fractions were greatly enriched in cytochrome c peroxidase activity for aerobically grown wild-type cultures, but petite and anaerobically grown wild-type cultures showed higher enzyme activities in supernatant fractions than was present in the corresponding particulate fractions after differential centrifugation. Evidence from low-temperature microspectroscopy, spectrophotometric assays of mitochondrial enzyme activities, and electron microscopy showed mitochondrial formation during the time required for preparation and lysis of spheroplasts from anaerobically grown cultures. The data were interpreted as indicating that cytochrome c peroxidase was an oxygen-inducible enzyme, and that there was a developmental relationship between enzyme-reactive membranes of mitochondria and cytoplasm during the period of respiratory adaptation.

Fine-structure analysis of intercellular and intracellular mitochondrial diversity in Saccharomyces cerevisiae.

Crosses were made between haploid wild-type and suppressive petite strains of bakers' yeast to obtain zygotes for analysis of mitochondrial heterogeneity. Wild-type x petite zygotes contained about 40% noncristate mitochondria when immediate mating mixtures were examined. The frequency of defective mitochondria had decreased to an average of 9.2% in 1-week-old zygote isolate cultures, and to 4.4% in slant cultures 1.5 years after initial zygote isolation. The latter value was not significantly different from values obtained with wild x wild zygotes of either age. The noncristate mitochondria were of two types: one lacking inner membrane invaginations or elaborations and the other containing concentrically arranged loops of inner membrane. The significance of these two types of respiration-deficient mitochondria is unknown. The gradual decrease in frequency of noncristate mitochondria, perhaps due to selection pressures in mixed chondriomes, was discussed as a further indication of the semiautonomous nature of the yeast organelle.