A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Preparation of highly phosphorylating mitochondria from the yeastSchizosaccharomyces pombe

Schizosaccharomyces pombe yeast cells grown on either fermentable or respiratory media were efficiently converted to stable spheroplasts by the -(1 3)-glucanase Novozym 234 in the presence of 1.2M sorbitol. Lysis of spheroplasts by gentle homogenization in dilute sorbitol resulted in the preparation of mitochondria with a structure similar to that observed within the starting yeast cells. The isolated mitochondria exhibited high oxidation rates with various respiratory substrates, NADH being the most efficient. The mitochondria appeared well coupled since the second State 4 rate observed after ADP consumption was identical to the initial one. The State 3 rate in the presence of ADP was completely inhibited by low oligomycin concentrations, similarly to the concomitant ATP synthesis of 900 nmol/min × mg protein. These NADH oxidation and dependent ATP-synthesis activities are much higher than those previously described for mitochondria isolated fromSchizosaccharomyces pombe, and similar to the highest values reported forSaccharomyces cerevisiae.     

Protein transport in the permeabilized cell of Schizosaccharomyces pombe.

We reconstituted a protein translocation-transport system composed of permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe and the precursor of alpha sex pheromone, prepro-alpha-factor of the budding yeast Saccharomyces cerevisiae. We found that P-cells prepared from the spheroplasts formed in 0.7M KCl as an osmotic stabilizer had the activity to transport pro-alpha-factor to the Golgi apparatus. Electron microscopic observations showed that membranes were preserved more intact in the P-cells prepared from the spheroplasts formed in 0.7M KCl than in 0.7M sorbitol. A glycoprotein of S. pombe contains galactose residues, and we detected incorporation of radiolabeled galactose residues into the anti-prepro-alpha-factor immunoprecipitable fractions in this S. pombe system, but not in the S. cerevisiae system. This paper reports that a heterologous system of in vitro protein transport was performed, and prepro-alpha-factor has the signals necessary for early steps of the transport in S. pombe.     

Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria

This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase??crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact??and alamethicin??permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca²⁺, (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca²⁺, (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca²⁺ and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.     

Involvement of the TOM complex in external NADH transport into yeast mitochondria depleted of mitochondrial porin1

The protein(s) responsible for metabolite transport through the outer membrane of the yeast Saccharomyces cerevisiae mitochondria depleted of mitochondrial porin (also known as voltage-dependent anion selective channel), termed here porin1, is (are) still unidentified. It is postulated that the transport may be supported by the protein import machinery of the outer membrane, the TOM complex (translocase of the outer membrane). We demonstrate here that in the absence of functional porin1, the blockage of the TOM complex by the fusion protein termed pb(2)-DHFR (consisting of the first 167 amino acids of yeast cytochrome b(2) preprotein connected to mouse dihydrofolate reductase) limits the access of external NADH to mitochondria. It was measured by the ability of the blockage to inhibit external NADH oxidation by the proper dehydrogenase located at the outer surface of the inner membrane. The inhibition depends on external NADH concentration and increases with decreasing amounts of the substrate. In the presence of 1 microg of pb(2)-DHFR per 50 microg of mitochondrial protein almost quantitative inhibition was observed when external NADH was applied at the concentration of 70 nmol per mg of mitochondrial protein. On the other hand, external NADH decreases the levels of pb(2)-DHFR binding at the trans site of the TOM complex in porin1-depleted mitochondria in a concentration-dependent fashion. Our data define an important role of the TOM complex in the transport of external NADH across the outer membrane of porin1-depleted mitochondria.     

NADH is specifically channeled through the mitochondrial porin channel in Saccharomyces cerevisiae

In many kinds of permeabilized cells, the restriction of metabolite diffusion by a mitochondrial porin closed state has been shown to control the respiration rate. However, since in isolated mitochondria the porin appears to be always open, the physiological relevance of a putative regulation via this channel status is now a subject of discussion. In Saccharomyces cerevisiae, in which some of the NADH dehydrogenase active sites are facing the intermembrane space, this regulatory mechanism might play an important role for the regulation of the cytosolic redox status. Using permeabilized spheroplasts from wild-type and porin-deficient mutant, we show that the NADH produced in the cytosol is channeled to the mitochondrial NADH dehydrogenases through a metabolic network involving the porin channel. Thus, the control exerted by the porin (i.e., open or closed state) seems to be determined through its participation or not in organized metabolic networks.     

Some Reactions of Isolated Corn Mitochondria Influenced by Juglone

The effects of juglone on the uptake of O₂ by excised corn roots (Zea mays L., Wf9 cms- T × M14) and isolated corn mitochondria arc reported. The O₂ uptake by excised corn roots, as measured by an O₂ electrode, was inhibited more than 90% after a one-hour treatment of 500 μM juglone. Lesser inhibitions were observed with 50 μM and 250 μM juglone. In a KC1 reaction medium in the absence of inorganic phosphate (Pi), juglone stimulated the rate of O₂ uptake by isolated mitochondria oxidizing NADH, succinate, or malate + pyruvate. In the presence of Pi, juglone concentrations of 3 μM and greater inhibited the state 3 oxidation rates of succinate and malate + pyruvate, lowered respiratory control and ADP/O ratios obtained from the oxidation of NADH, malate + pyruvate, or succinate, and reduced the coupled deposition of calcium phosphate within isolated mitochondria driven, by the oxidation of malate + pyruvate. The inhibition of state 3 O₂ uptake by isolated mitochondria, an oxidative state in which electron transfer is coupled to ATP production, is seen to correlate with the inhibition affected by juglone when applied to tissues in vivo.     

Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces Cerevisiae

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effects of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is disscussed.     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Isolation and Oxidative Properties of Intact Mitochondria from the Leaves of Sedum praealtum: A Crassulacean Acid Metabolism Plant

A procedure is described for preparing intact mitochondria from leaves of Sedum praealtum D.C., a plant showing Crassulacean acid metabolism. These mitochondria oxidized malate, pyruvate, α-ketoglutarate, succinate, NADH, NADPH, and isocitrate with good respiratory control and ADP/O ratios better than those observed in mitochondria from other photosynthetic tissues.     

Properties of substantially chlorophyll-free pea leaf mitochondria prepared by sucrose density gradient separation

Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, α-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca²⁺, whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O₂ uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase.     

Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes

At the pyruvate branch point, the fermentative and oxidative metabolic routes diverge. Pyruvate can be transformed either into lactate in mammalian cells or into ethanol in yeast, or transported into mitochondria to fuel ATP production by oxidative phosphorylation. The recently discovered mitochondrial pyruvate carrier (MPC), encoded by MPC1, MPC2, and MPC3 in yeast, is required for uptake of pyruvate into the organelle. Here, we show that while expression of Mpc1 is not dependent on the carbon source, expression of Mpc2 and Mpc3 is specific to fermentative or respiratory conditions, respectively. This gives rise to two alternative carrier complexes that we have termed MPC_FERM and MPC_OX. By constitutively expressing the two alternative complexes in yeast deleted for all three endogenous genes, we show that MPC_OX has a higher transport activity than MPC_FERM, which is dependent on the C‐terminus of Mpc3. We propose that the alternative MPC subunit expression in yeast provides a way of adapting cellular metabolism to the nutrient availability.     

Acidic cytochrome c6 of unicellular cyanobacteria is an indispensable and kinetically competent electron donor to cytochrome oxidase in plasma and thylakoid membranes.

Cytochromes c6 from three cyanobacteria were tested as substrates for membranous cyt. c oxidase(aa3) of Anacystis and Synechocystis using intact spheroplasts or isolated plasma(CM) and thylakoid(ICM) membranes. Neither spheroplasts nor CM/ICM gave significant O2 uptake rates with NADH without added cyt. c. Horse cyt. c (at low ionic strength) or cyt. c6 from Anacystis, Synechocystis or Microcystis (at high ionic strength) supported substantial HCN- & CO-sensitive NADH oxidase activity, consistent with in vivo O2 uptake. Cyanobacterial respiratory electron transport involves NADH dehydrogenase(fpN), plastoquinone, cyt. b/c(f), cyt. c6 & cyt. aa3, in both CM & ICM. In ICM, fpN and cyt. aa3 are functionally replaced in the light by PS II and PS I, respectively. In both membranes, cyt. c6 is an obligatory electron donor to cyt. aa3 &/or to P700. Respiratory action of acidic cyt. c6 (in unicellular species) may be unmasked only under conditions of elevated ionic strength.     

The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F₁F₀-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K_0.5 for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.

3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c₁ (552 nm) and c (545 and 516 nm).

4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.

5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.

6. The responses of yeast mitochondria to Ca²⁺ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate.     

Porin and cytochrome oxidase containing contact sites involved in the oxidation of cytosolic NADH

Cytochrome c (cyto-c) added to isolated mitochondria promotes the oxidation of extra-mitochondrial NADH and the reduction of molecular oxygen associated to the generation of an electrochemical membrane potential available for ATP synthesis. The electron transport pathway activated by exogenous cyto-c molecules is completely distinct from the one catalyzed by the respiratory chain. Dextran sulfate (500 kDa), known to interact with porin (the voltage-dependent anion channel), other than to inhibit the release of ATP synthesized inside the mitochondria, greatly decreases the activity of exogenous NADH/cyto-c system of intact mitochondria but has no effect on the reconstituted system made of mitoplasts and external membrane preparations. The results obtained are consistent with the existence of specific contact sites containing cytochrome oxidase and porin, as components of the inner and the outer membrane respectively, involved in the oxidation of cytosolic NADH. The proposal is put forward that the bi-trans-membrane electron transport chain activated by cytosolic cyto-c becomes, in physio-pathological conditions: (i) functional in removing the excess of cytosolic NADH; (ii) essential for cell survival in the presence of an impairment of the first three respiratory complexes; and (iii) an additional source of energy at the beginning of apoptosis.     

Transport of proteins across the mitochondrial outer membrane. A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase.

Cytochrome c peroxidase, a cytoplasmically made enzyme located between the inner and outer membrane of yeast mitochondria, is synthesized as larger precursor in a reticulocyte cell-free lysate as well as in pulsed yeast spheroplasts. When the pulsed spheroplasts are chased, the precursor is converted to the mature apoprotein. When the in vitro synthesized precursor is incubated with isolated yeast mitochondria in the absence of protein synthesis, it is cleaved to the mature form; the mature form co-sediments with the mitochondria and is resistant to externally added proteases. These results, in conjunction with those reported earlier (Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347) suggest that the mechanism of protein transport into the mitochondrial intermembrane space is quite similar to that of protein transport into the matrix or the inner membrane.     

Respiration Rate and Mitochondrial Activity in Iris Bulbs

The oxygen uptake of iris bulbs (Iris×hollandica‘Wedgwood’) which had been stored dry at 30 C (“retarded’ bulbs) was low (10 μmol O₂ per h and bulb), the oxygen uptake of the intact bulb, the three outer fleshy scales and the remaining central part of the bulb increased three- to fourfold, nearly twofold and fourfold, respectively. Mitochondria were isolated from the scales of retarded and activated bulbs and their oxygen consumption with succinate, l -malate (plus pyruvate). x-ketoglutarate and NADH as substrate was measured polarographically. The oxidative capacity of mitochondria isolated from the scales of activated bulbs was only slightly higher than that from retarded bulbs when calculated on a tissue basis. No difference was found between the phosphorylation efficiency, respiratory control, cytochrome c deficiency, succinate dehydrogenase, malate dehydrogenase, succinate-cytochrome c rductase, NADH-cytochrome c reductase and cytochrome oxidase activity of the retarded and activated bulbs. The increase in the in vitro oxygen uptake of the scales after transition from 30 to 13 C was not accompanied by an equal increase in the oxidative capacity of their mitochondria suggesting that they are not responsible for this rise in oxygen uptake.     

Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from <Emphasis Type="Italic">Kalanchoë pinnata</Emphasis>

Kalanchoë pinnata mitochondria readily oxidized succinate, malate, NADH, and NADPH at high rates and coupling. The highest respiration rates usually were observed in the presence of succinate. The high rate of malate oxidation was observed at pH 6.8 with thiamine pyrophosphate where both malic enzyme (ME) and pyruvate dehydrogenase were activated. In CAM phase III of K. pinnata mitochondria, both ME and malate dehydrogenase (MDH) simultaneously contributed to metabolism of malate. However, ME played a main function: malate was oxidized via ME to produce pyruvate and CO₂ rather than via MDH to produce oxalacetate (OAA). Cooperative oxidation of two or three substrates was accompanied with the dramatic increase in the total respiration rates. Our results showed that the alternative (Alt) pathway was more active in malate oxidation at pH 6.8 with CoA and NAD⁺ where ME operated and was stimulated, indicating that both ME and Alt pathway were related to malate decarboxylation during the light. In K. pinnata mitochondria, NADH and NADPH oxidations were more sensitive with KCN than that with succinate and malate oxidations, suggesting that these oxidations were engaged to cytochrome pathway rather than to Alt pathway and these capacities would be desirable to supply enough energy for cytosol pyruvate orthophosphate dikinase activity.