Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor.

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown "petite-negative" yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40 degrees C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles. 2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerol-grown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae. 3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the "petite-positive" yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126. 4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 muM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 muM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor. 5. We conclude that "petite-positive" and "petite-negative" yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Subunit composition of RNA polymerase II from the fission yeast Schizosaccharomyces pombe

The subunit composition of RNA polymerase II (polII) was compared between the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. For this purpose, we partially purified the enzyme from S. pombe. Judging from the co-elution profiles in column chromatographies of both the RNA polymerase activity and the two large subunit polypeptides (subunit 1 (prokaryotic β' homologue) and subunit 2 (β homologue)), the minimum number of S. pombe polII-associated polypeptides was estimated to be ten, less than the proposed subunit number of the S. cerevisiae enzyme. These ten putative subunits of S. pombe polII correspond to subunits 1, 2, 3, 5, 6, 7, 8, 10, 11 and 12 of the S. cerevisiae counterparts     

Bongkrekic acid sensitivity of respiration-deficient mutants and of petite-negative species of yeasts

1. Growth on glucose of cytoplasmic respiration-deficient (ρ⁻) mutants isolated from five strains of Saccharomyces cerevisiae and one strain of Saccharomyces carlsbergensis were arrested by the inhibitor of mitochondrial adenine nucleotide translocation, bongkrekic acid. This indicates that the mitochondrial adenine nucleotide translocation system is preserved and necessary for growth in a number of independent ρ⁻ mutants.

2. Growth of three “petite-negative” yeast species was arrested by a combined inhibition of respiration by antimycin A and of adenine nucleotide translocation by bongkrekic acid. Thus, the arrest of growth upon inhibition of adenine nucleotide translocation in non-respiring cells is not specific for ρ⁻ mutants and may be a general characteristic of eucaryotic cells.     

Respiration-dependent uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitochondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATPase activity was stimulated at higher concentrations of uncouplers.

2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response.

3. The addition of succinate, NADH or ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin.

4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10^-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin.

5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity.

6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.     

Diphosphatidylglycerol-induced changes in the organization of mitochondrial ATPase

1. On submitochondrial particles from bovine heart, diphosphatidylglycerol produced a selective solubilization of ATPase. The solubilized enzyme was purified further by ammonium sulfate fractionation and shown to have the same reconstitutive activity as coupling factor F₁ (Pullman, M.E., Penefsky, H. S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322–3329).

2. Diphosphatidylglycerol-treated submitochondrial particles retained large amounts of the phospholipid and showed a decreased ATPase activity. Once the excess of phospholipid was removed, soluble ATPase could be again reincorporated in an oligomycin-sensitive complex.

3. On Mg-ATP particles the solubilization of ATPase induced by diphosphatidylglycerol was preceded by a stimulation of oligomycin-sensitive ATPase which indicated a dissociation of F₁ from the ATPase inhibitor (Pullman, M. E. and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762–3769). Magnesium was required to obtain the oligomycin-sensitive stimulation whereas in the absence of magnesium the solubilization of ATPase was prevalent.

4. It is concluded that the decreased association of F₁ with the ATPase inhibitor produced by diphosphatidylglycerol, causes a labilization of ATPase-membrane interaction. Under particular conditions, e.g. a high amount of phospholipid and a low concentration of magnesium, this is followed by the detachment of ATPase.     

Proteins required for the binding of mitochondrial ATPase to the mitochondrial inner membrane

1. Isolated F₁ (mitochondrial ATPase) binds to urea-treated submitochondrial particles suspended in sucrose/Tris/EDTA with a dissociation constant of 0.1 μM.

2. About one-third of the F₁ and the oligomycin-sensitivity conferring protein (OSCP) are lost during preparation of submitochondrial particles prepared at high pH (A particles). None is lost from particles treated with trypsin (T particles).

3. After further treatment with alkali of urea-treated particles, binding of F₁ requires the addition of OSCP. Maximum binding is reached when both OSCP and Fc₂ are added. The concentration of F₁-binding sites in the presence of both OSCP and Fc₂ is about the same as that in TU particles.

4. After further extraction with silicotungstate of urea- and alkali-treated particles, OSCP no longer induces binding of F₁, unless Fc₂ is also present. Fc₂ induces binding in the absence of OSCP but with a lower binding constant and, in contrast to results under all the other conditions studied in this paper, the ATPase activity is oligomycin insensitive.

5. It is tentatively concluded that OSCP is the binding site for F₁ and Fc₂ is the binding site for OSCP.     

Identification of novel suppressors for Mog1 implies its involvement in RNA metabolism, lipid metabolism and signal transduction

Mog1 is conserved from yeast to mammal, but its function is obscure. We isolated yeast genes that rescued a temperature-sensitive death of S. cerevisiae Scmog1Δ, and of S. pombe Spmog1^ts. Scmog1Δ was rescued by Opi3p, a phospholipid N-methyltransferase, in addition to S. cerevisiae Ran-homologue Gsp1p, and a RanGDP binding protein Ntf2p. On the other hand, Spmog1^ts was rescued by Cid13 that is a poly (A) polymerase specific for suc22⁺ mRNA encoding a subunit of ribonucleotide reductase, Ssp1 that is a protein kinase involved in stress response pathway, and Crp79 that is required for mRNA export, in addition to Spi1, S. pombe Ran-homologue, and Nxt2, S. pombe homologue of Ntf2p. Consistent with the identification of those suppressors, lack of ScMog1p dislocates Opi3p from the nuclear membrane and all of Spmog1^ts showed the nuclear accumulation of mRNA. Furthermore, SpMog1 was co-precipitated with Nxt2 and Cid13.     

Characterization of a mutant of Schizosaccharomyces pombe lacking cytochrome b-566

1. A chromosomal respiration-deficient mutant of the petite-negative yeast Schizosaccharomyces pombe was isolated. Its mitochondria show respiration rates of about 7% of the wild-type respiration with NADH and succinate as substrate, and 45% with ascorbate in the presence of tetramethyl-p-phenylenediamine. Oxidation of NADH and succinate is insensitive to antimycin and cyanide and that of ascorbate is much less sensitive to cyanide than the wild type.

2. The amounts of cytochromes c₁ and aa₃ are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.

3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH₂-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.

4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.

5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.

6. Cytochromes aa₃, c and c₁ are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.

7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

EPR studies on the respiratory chain of wild-type Saccharomyces cerevisiae and mutants with a deficiency in succinate dehydrogenase

1. Three nuclear mutants of Saccharomyces cerevisiae deficient in succinate dehydrogenase have been isolated. Two of these mutants are allelic.

2. The amount of covalently bound flavin of submitochondrial particles of the two allelic mutants is about 14% and that of the third mutant about 50% of the amount in wild-type particles. The turnover number of succinate dehydrogenase of particles is decreased in all mutants. The turnover number of fumarate reductase is increased in the two allelic mutants, but decreased in the third mutant.

3. EPR spectra, measured at 82 °K, show that the amplitude of the g = 1.93 signal in particles of the two allelic mutants is less than 10% of that in wild-type particles. It is concluded that iron-sulphur centres other than those of succinate dehydrogenase make only a negligible contribution to the line at g = 1.93 in wild-type particles.

4. EPR measurements below 20 °K show that the amplitude of the signal at g = 2.01 detected in oxidized particles is decreased in particles of the two allelic mutants.

5. A signal with lines at g = 2.027 and g = 1.933 is detected at low temperatures in all particle preparations, even in those from a cytoplasmic petite mutant. It is suggested that this signal is derived from a contaminant and not from the inner membrane.     

Regulation of primary carbon metabolism in Kluyveromyces lactis

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called “Nonconventional Yeasts.” The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the “cell factory” concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.     

Association and dissociation of the thick and thin filaments within myofibrils in conditions of “contraction” and “relaxation”

1. The current assumption that the low ATPase activity of relaxed myofibrils is represented by the ATPase activity of myosin which has been set free during the dissociation of actomyosin was investigated. For this purpose, the ATPase activity of relaxed skeletal myofibrils of the rabbit and of the crab Maia squinado has been compared with the activity of contracted fibrils and of purified rabbit myosin in conditions of varying ionic strength, pH and concentrations of MgATP (i.e. MgATP²⁻ + MgHATP⁻) and Mg²⁺.

2. Contraction and relaxation of the fibrils was induced by changing the concentration of Ca²⁺ from about 5×10⁻⁵ to below 1×10⁻⁸ M.

3. In all conditions studied, the ATPase activity of relaxed fibrils was about 6–8 times less than that of the contracted fibrils, but it remained a typical actomyosin ATPase.

4. Quantitatively and qualitatively, this ATPase differs from the ATPase of myosin. For instance, its dependence on pH is the reverse of that of the myosin ATPase.

5. Calculation showed that the fibrils are dissociated by 90% in conditions of relaxation. Since the ATPase activity of myosin was merely some 2% of the actomyosin activity, the major part of the ATPase of fibrils, even at a dissociation of 90%, is bound to show the properties of the ATPase of actomyosin.

6. However, a dissociation of 90% cannot be distinguished from a dissociation of 100% by means of physical methods (viscosity, superprecipitation, resistance to stretch, etc.). This explains why physical methods indicate a “full” dissociation of actomyosin although, enzymatically, the ATPase is still of the actomyosin type.

7. The possible reasons are discussed for the discrepancy between the 100-fold increase in the ATP turnover and the 1000-fold increase in energy turnover of the living muscle during the transition from relaxed to active state. The most probable explanation seems to be an ATPase activity of myosin which is too high by a factor of ten as compared to the energy turnover of living muscle at the resting state. This high activity cannot be caused by a contamination of the myosin by Ca²⁺-insensitive actomyosin.     

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg²⁺ dependent; Co²⁺ and Mn²⁺ but not Ca²⁺ could replace Mg²⁺ to some extent; the activation by Mg²⁺ was slightly antagonized by Ca²⁺. Even in the presence of Mg²⁺, Na⁺ or K⁺ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]_{0.5 V} = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg²⁺. Solubilization, however, led to instability of the enzyme.

The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10^-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN₃ had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.

Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H⁺ translocation. A H⁺-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H⁺-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

Studies on the activation of purified mitochondrial ATPase by phospholipids

1. An ATPase which is activated by phospholipids and inhibited by oligomycin, has been purified from beef heart submitochondrial particles using affinity chromatography. Phospholipid and detergent are removed by washing the enzyme with a solution of serum albumin while it is attached to the biospecific adsorbent.

2. The ATPase is activated up to 18-fold by lysolecithin and to a smaller extent by cardiolipin, phosphatidylinositol and phosphatidylethanolamine. The amount required of each of these phospholipids to give half-maximal activation is apparently inversely related to the number of fatty acid chains in the lipid. Lecithin, which is a poor activator of the ATPase, competitively inhibits the activation by cardiolipin.

3. The activation of the ATPase consists of an increase in both the maximal velocity of the reaction and the affinity for substrate ATP. The pH optimum of the reaction is not influenced by the charge of the lipid.

4. Arrhenius plots of ATPase activated with lysolecithin show a transition to a higher activation energy at temperatures below 19 °C. The sensitivity of the lysolecithin-activated enzyme to oligomycin is markedly reduced below the same temperature. With cardiolipin the transition is observed at 13 °C.

5. ADP, Mg²⁺ and to a smaller extent ATP, Mg²⁺ enhance the activation of ATPase by suboptimal amounts of phospholipid.     

Studies on the mechanism of inhibition of the mitochondrial electron transport by antimycin. II. Antimycin as an allosteric inhibitor

1. Pretreatment of sub-mitochondrial particles with cholate results in a change in the curve describing inhibition by antimycin of the succinate-cytochrome c reductase from sigmoidal towards linear. This effect of cholate is reversed by partial removal of the cholate by dialysis, either in the absence or presence of antimycin.

2. Treatment with cholate has the same action on the sigmoidal effect curve of antimycin on the reducibility of cytochrome b. This is also reversed by dialysis.

3. The effect of antimycin on the displacement to the red of the -band of ferrocytochrome b, measured in the presence of succinate, NADH or reduced ubiquinone Q-2, is also described by a sigmoidal curve that is changed to a linear one by addition of cholate.

4. Linear displacement curves are obtained with menaquinol or Na₂S₂O₄.

5. It is proposed that antimycin is an allosteric inhibitor of the respiratory chain. This allosteric effect should be distinguished from the effect of antimycin on the “conformation stability” of Complex III.     

Extraction of mitochondrial protein-lipid complexes into organic solvents: an approach to study the interaction between the ATPase and the mitochondrial ATPase-inhibitor protein

Protein-lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein-lipid residue left after solvent removal from these extracts was used to form liposomes which display low-temperature-resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1-ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4--10-fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase-inhibitor protein and (F1--ATPase)-inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site.     

Oxidative phosphorylation in yeast VI. ATPase activity and protein synthesis in mitochondria isolated from nuclear mutants deficient in cytochromes

1. The oligomycin-sensitive Mg²⁺-dependent ATPase activity of mitochondria isolated from wild-type yeast Saccharomyces cerevisiae was only slightly inhibited by atractyloside at concentrations which entirely prevented oxidative phosphorylation. This indicated that most of the ATPase in these mitochondrial preparations was located outside the atractyloside-sensitive barrier and did not participate in the energy-transfer process.

2. ATPase activity of mitochondria isolated from nuclear gene mutants deficient in a single cytochrome, a, b, or c, respectively, was strongly inhibited by oligomycin. The mitochondria from these mutants, like those from the wild-type strain, were able to incorporate amino acids into protein.

3. Mitochondrial ATPase activity of single nuclear gene mutants deficient in both cytochromes a and b was only slightly inhibited by oligomycin. These mitochondria were incapable of incorporating amino acids into protein. The mitochondria from these nuclear mutants thus resembled mitochondria of cytoplasmic respiration-deficient mutants.

4. The results suggest that mitochondrial cytochromes may be coded by nuclear genes and that product(s) of mitochondrial protein synthesis may be required for integrating the cytochromes a and b and the components of the oligomycin-sensitive ATPase complex into the mitochondrial membranes.     

Drad21, a Drosophila rad21 homologue expressed in S-phase cells

Cohesin is an evolutionarily conserved multiprotein complex required to establish and maintain sister chromatid cohesion. Here, we report the cloning and initial characterization of the Drosophila homologue of the fission yeast rad21 cohesin subunit, called Drad21. The Drad21 coding region was localized to centromeric heterochromatin and encodes a 715 amino acid (aa) protein with 42% aa identity to vertebrate Rad21p-homologues, 25% with Scc1p/Mcd1p (S. cerevisiae) and 28% with Rad21p (S. pombe). Sequences with similarity to the sites of proteolytic cleavage identified in Scc1p/Mcd1p are not evident in DRAD21. Northern blot and mRNA in-situ studies show that Drad21 is developmentally regulated, with high levels of expression in early embryogenesis, in S-phase cells of proliferating imaginal tissues, and in the early endocycling cells of the embryonic gut.     

Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C.W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35–43].