James R. Mattoon

Ca2+ metabolism in yeast cells and mitochondria

Abstract 1. 1. Ca 2+ in the culture medium stimulates only slightly the growth and respiration of Saccharomyces cerevisiae . 2. 2. Neither energy-linked Ca 2+ transport nor high-affinity Ca 2+ binding occur in mitochondria isolated from Saccharomyces cerevisiae or from Torulopsis utilis . 3. 3. Metabolism-independent, low-affinity binding of Ca 2+ does, however, occur in mitochondria isolated from both yeasts. The concentration of these sites varies between 40 and 50 nmoles per mg of mitochondrial protein. Their affinity for Ca 2+ is rather low ( K m = 10–20 μ M). 4. 4. Mitochondria of Saccharomyces cerevisiae contain about 10 nmoles of endogenous Ca 2+ per mg of mitochondrial protein, which is bound or sequestered in a very stable manner. 5. 5. The respiration of mitochondria from Saccharomyces cerevisiae is stimulated by valinomycin in the presence of KCl, suggesting that energy-linked transport of K + occurs. 6. 6. The results show that energy-linked Ca 2+ transport is not a universal attribute of intact mitochondria from all species. Since the yeast cells lack high-affinity Ca 2+ binding capacity, they may lack a specific Ca 2+ carrier. The basic energy-dependent cation pump may however be present in these mitochondria, since they can transport K + in the presence of valinomycin.

Regulation of mitochondrial biogenesis : enzymatic changes in cytochrome-deficient yeast mutants requiring delta-aminolevulinic acid.

Yeast cells almost completely deficient in all cytochromes were obtained by introducing two defective nuclear genes, cyd1 and cyc4, into the same haploid strain. The action of the two mutant genes is synergistic, since either gene acting singly results in only partial cytochrome deficiency. Normal synthesis of all cytochromes can be restored in the double mutant by adding delta-aminolevulinic acid to the growth medium. The optimum concentration of delta-aminolevulinate for restoration of cytochrome synthesis is about 40 muM; when higher concentrations are used, synthesis of cytochromes is partially suppressed, particularly that of cytochrome a.a3. Growth yield of the double mutant is stimulated by ergosterol and Tween 80, a source of unsaturated fatty acid. Methionine stimulates further. None of these nutrients is required for growth when sufficient delta-aminolevulinic acid is present in the growth medium. With respect to nutritional responses, the single-gene, cytochrome-deficient mutant, ole3, behaves like the double mutant. The frequency of the p-mutation in the double mutant grown in the absence of ergosterol, Tween 80, and delta-aminolevulinic acid is at least 15%. The frequency can be reduced to less than 1% by either delta-aminolevulinic acid or Tween 80. Ergosterol alone does not decrease the p- frequency. The ole3 mutant does not exhibit increased p-frequency under similar conditions of unsaturated fatty acid deficiency.

Oligomycin resistance in normal and mutant yeast

Abstract Yeast mutants with genetically altered mitochondria are extremely useful in the study of oxidative phosphorylation mechanisms ( Beck et al. , 1968 ). Such mutants are usually detected by their inability to grow on non-fermentable carbon sources, their spectral characteristics, and by selective staining. Drug resistance has also been employed in obtaining mitochondrial mutants ( Thomas and Wilkie, 1968 ; Butow and Zeydel, 1968 ), but as yet this method has not been extended to include specific inhibitors of oxidative phosphorylation. This report describes the isolation of 50 oligomycin-resistant Saccharomyces cerevisiae mutants and the genetic characterization of two of these mutants and a naturally resistant strain. At least two Mendelian genes control the resistance in these strains. Resistance displays partial dominance and additivity in heterozygous diploids. Oligomycin partially inhibits cytochrome synthesis, and increases the frequency of mutation to cytoplasmic respiratory deficiency (p − ). Oligomycin resistance is accompanied by resistance to venturicidin.

Cation movements and respiratory response in yeast mitochondria treated with high Ca2+ concentrations

Abstract 1. 1. Reinvestigation of the interaction of Ca 2+ with mitochondria isolated from the yeasts Saccharomyces cerevisiae and Candida utilis has shown that respiration-linked cation uptake can be induced by high Ca 2+ concentrations, in the range of 1–10 mM. 2. 2. High Ca 2+ concentration induces H + ejection, 2-fold respiratory stimulation, and an increase in the steady-state oxidation level of cytochrome b . 3. 3. Ca 2+ uptake by yeast mitochondria was demonstrated by spectrophometric recording of changes in calcium-murexide absorption. 4. 4. Both Ca 2+ uptake and proton ejection are strongly inhibited by uncouplers and by the electron transport inhibitor, antimycin A. 5. 5. Inorganic phosphate stimulates the initial Ca 2+ uptake rate about 8-fold. 6. 6. No appreciable ATP-driven Ca 2+ uptake could be detected under conditions suitable for respiration-linked transport. 7. 7. Respiration-linked cation transport by yeast mitochondria has a narrow specificity, similar to that of liver mitochondria; Ca 2+ , Sr 2+ and Mn 2+ are active, while Mg 2+ and Na + are not. 8. 8. Although isolated yeast mitochondria have the capacity for specific, respiration-linked Ca 2+ uptake, this is probably of little physiological significance, since an efficient, high affinity Ca 2+ transport system is lacking.

[26] Yeast mitochondria and submitochondrial particles

Publisher Summary 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.

Nucleo-cytoplasmic interaction between oligomycin-resistant mutations in Saccharomyces cerevisiae

Summary1.A single-gene nuclear mutant of Saccharomyces cerevisiae, isolated as oligomycin-resistant, exhibits in vivo cross-resistance to venturicidin and collateral sensitivity to Synthalin. All three compounds are inhibitors of mitochondrial oxidative phosphorylation. Oligomycin resistance and Synthalin sensitivity are recessive, while venturicidin resistance is dominant.2.A cytoplasmic mutant, also isolated as oligomycin-resistant, shows collateral sensitivity to both Synthalin and venturicidin. All three traits undergo mitotic segregation in diploids formed by crossing mutant and normal haploids.3.A novel nucleocytoplasmic interaction is observed in diploids formed by crossing haploid strains containing the nuclear and the cytoplasmic mutations, respectively. The dominant venturicidin resistance determined by the nuclear gene undergoes mitotic segregation, which results from a suppression of the nuclear phenotype by the cytoplasmic mutation. When a diploid mitotic segregant contains primarily mutant-type mitochondria, venturicidin resistance is completely suppressed. In haploids containing both the nuclear and cytoplasmic mutations, suppression is only partial.4.Oxidative phosphorylation and ATPase in mitochondrial fractions isolated from cytoplasmic mutant cells are less sensitive to inhibition by oligomycin than normal, but in vitro sensitivity to venturicidin is not significantly changed. In similar mitochondrial fractions isolated from normal and nuclear mutant cells, no significant differences in sensitivity to either inhibitor are detected.5.The molecular basis for the nucleocytoplasmic suppression of venturicidin resistance may involve participation of mitochondrial membrane, plasma membrane or both. Either mitochondria can undergo changes in venturicidin sensitivity upon isolation, or the molecular entity which controls access of venturicidin to the mitochondria resides outside of the organelles.6.Our data establish that aspects of the response in vivo of both venturicidin and Synthalin are controlled by the mitochondrial genome.7.The nucleocytoplasmic interaction described here is the first example in which a specific restricted mitochondrial mutation modifies the phenotypic expression of a nuclear gene.

Cryobiological studies of yeast mitochondria

Summary Mitochondria from the yeast S. cerevisiae have been shown to be particularly suited for cryobiological studies, since they may be frozen and thawed without detectable injury to either structure or function. Mitochondria are frozen in the usual isolation medium containing mannitol and serum albumin, and no penetrating cryoprotective agent such as dimethyl sulfoxide or glycerol is required. Yeast cells have been stored at 4°C for at least 9 days, and isolated mitochondria for 30 days or more, in liquid N2 without appreciable change in mitochondrial Qo2, ADP:O, or adenosine diphosphate-linked respiratory control. Stored mitochondria retain normal morphology and capacity for adenine nucleotide translocation. Respiratory control has been preserved for as long as 53 days. Improved isolation methods together with these storage techniques have permitted study of intact yeast mitochondria with a facility virtually unattainable with mitochondria from other cell types. Mitochondria isolated from a yeast mutant with deficient aerobic energy metabolism exhibit increased lability during freezing and thawing. Both Qo2 and respiratory control are greatly reduced.

A rapid semimicro method for production of yeast mitochondria

Abstract A simple, rapid, cconomical method for the production of yeast mitochondria is deseribed in detail. Highly intact mitochondria are isolated from cells mechanically ruptured with a small colloid mill. Yields of about 10 mg mitochondrial protein per 30 gm cells routinely are obtained. Because enzymic pretreatment of cells is unnecessary, intact mitochondria can be prepared from both parent and mutant cell strains in various physiological states using a standard set of conditions.

Colorimetric determination of succinic acid using yeast succinate dehydrogenase

An enzymatic method for the rapid determination of succinic acid in biological fluids was developed utilizing yeast mitochondria as a source of succinate dehydrogenase. The yeast enzyme catalyzes a complete stoichiometric reduction of 2- (p-iodophenyl)-3-(p-nitrophenyl)-5-tetrazolium chloride to a red formazan. The formazan is extracted into ethylacetate and its absorbance measured at 490 nm. The method is simple, specific, reproducible, and very sensitive (0.01 to 0.14 mumol). The yeast enzyme can be stored in liquid nitrogen for periods of at least 30 days with no significant change in specific activity. In this respect it is superior to a variety of succinate dehydrogenase preparations from animal tissues. The method was applied to measurement of succinic acid excreted by nonproliferating yeast cells metabolizing glucose. Derepressed yeast cells secreted several-fold as much succinic acid as repressed cells submitted to identical test conditions.

Effect of filipin on Rat-liver and yeast mitochondria

Abstract 1. Under the appropriate conditions intact yeast and mammalian mitochondria exhibit a heretofore unobserved sensitivity to the polyene antibiotic, filipin. The activity of the “filipin complex” (Filipins I, II, III and IV) is shown to be primarily due to the component designated Filipin II. 2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited α-ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate. 3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg2+, K+, Ca2+ or 2,4-dinitrophenol, but is reversed by cytochrome c. 4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes.