Effect of cytochrome oxidase inhibitors on the yeast thermotolerance

The investigation of the effect of the cytochrome oxidase inhibitors sodium cyanide and sodium azide on the thermotolerance of the yeasts Rhodotorula rubra, Debaryomyces vanriji, and Saccharomyces cerevisiae showed that these inhibitors diminish the thermotolerance of R. rubra and D. vanriji, but do not affect the thermotolerance of S. cerevisiae. Taking into account the fact that, unlike the latter yeast, R. rubra and D. vanriji are nonfermentative yeasts, the difference in the effects of the inhibitors on the yeast thermotolerance can be readily explained by the different types of glucose utilization (either oxidative or fermentative) in these yeasts. The data obtained also provide evidence that there is a correlation between the functional activity of mitochondria and the thermotolerance of yeast cells.     

Regulation of the biosynthesis of cytochrome P-450 in brewer's yeast. Role of cyclic AMP.

The drug metabolising enzyme cytochrome P-450 has been studied in great detail in mammalian systems and its presence in microorganisms is also well established. However, neither its function nor its means of control in brewer's yeast, Saccharomyces cerevisiae, has been investigated. We demonstrate here using yeast protoplasts that it is the intracellular concentration of cyclic AMP which controls, by repression, the de novo synthesis of the enzyme, and also that cyclic AMP concentrations are in turn inversely related to the concentration of glucose in the yeast growth medium.     

Level of cyclic AMP during induction of alternative oxidase in Yarrowia lipolytica yeast cells

The effect of cyanide, antimycin A, ethanol, and acetate on the induction of alternative oxidase in the yeast Yarrowia lipolytica VKM Y-155 sensitive to cyanide, in the presence of the aforementioned compounds led to the development of cyanide-resistant respiration, which could be suppressed by benzohydroxamic acid, an inhibitor of alternative oxidases. The incubation of cells with cyanide, ethanol, or acetate raised the intracellular pool of cAMP, which attained maximal values after a 2- to 3-min incubation, then rapidly decreased to the initial value and did not change over the next three hours of incubation. The possible role of cAMP in the induction of alternative oxidase in yeast cells is discussed.     

Regulation of the biosynthesis of cytochrome P-450 in brewer's yeast role of cyclic AMP

The drug metabolising enzyme cytochrome P-450 has been studied in great detail in mammalian systems and its presence in microorganisms is also well established. However, neither its function nor its means of control in brewer's yeast, Saccharomyces cerevisiae, has been investigated. We demonstrate here using yeast protoplasts that it is the intracellular concentration of cyclic AMP which controls, by repression, the de novo synthesis of the enzyme, and also that cyclic AMP concentrations are in turn inversely related to the concentration of glucose in the yeast growth medium.     

Role of cyclic AMP in mitochondriogenesis in yeast

Cyclic AMP prevents the release of proteins from mitochondria (prelabelled with radioactive aminoacids) during glucose repression in yeast cells and chloramphenicol has no effect on this process. Using specific antisera, the release of the mitochondrially synthesised membrane factor of ATPase during repression and the blocking of this by cAMP has been demonstrated.     

Modular assembly of yeast cytochrome oxidase

Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high–molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.     

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis

Eukaryotic cytochrome c oxidase (CcO), the terminal enzyme of the energy-transducing mitochondrial electron transport chain is a hetero-oligomeric, heme–copper oxidase complex composed of both mitochondrially and nuclear-encoded subunits. It is embedded in the inner mitochondrial membrane where it couples the transfer of electrons from reduced cytochrome c to molecular oxygen with vectorial proton translocation across the membrane. The biogenesis of CcO is a complicated sequential process that requires numerous specific accessory proteins, so-called assembly factors, which include translational activators, translocases, molecular chaperones, copper metallochaperones and heme a biosynthetic enzymes. Besides these CcO-specific protein factors, the correct biogenesis of CcO requires an even greater number of proteins with much broader substrate specificities. Indeed, growing evidence indicates that mitochondrial ATP-dependent proteases might play an important role in CcO biogenesis. Out of the four identified energy-dependent mitochondrial proteases, three were shown to be directly involved in proteolysis of CcO subunits. In addition to their well-established protein-quality control function these oligomeric proteolytic complexes with chaperone-like activities may function as molecular chaperones promoting productive folding and assembly of subunit proteins. In this review, we summarize the current knowledge of the functional involvement of eukaryotic CcO-specific assembly factors and highlight the possible significance for CcO biogenesis of mitochondrial ATP-dependent proteases.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Coa2 is an assembly factor for yeast cytochrome c oxidase biogenesis that facilitates the maturation of Cox1

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is dependent on a new assembly factor designated Coa2. Coa2 was identified from its ability to suppress the respiratory deficiency of coa1Delta and shy1Delta cells. Coa1 and Shy1 function at an early step in maturation of the Cox1 subunit of CcO. Coa2 functions downstream of the Mss51-Coa1 step in Cox1 maturation and likely concurrent with the Shy1-related heme a(3) insertion into Cox1. Coa2 interacts with Shy1. Cells lacking Coa2 show a rapid degradation of newly synthesized Cox1. Rapid Cox1 proteolysis also occurs in shy1Delta cells, suggesting that in the absence of Coa2 or Shy1, Cox1 forms an unstable conformer. Overexpression of Cox10 or Cox5a and Cox6 or attenuation of the proteolytic activity of the m-AAA protease partially restores respiration in coa2Delta cells. The matrix-localized Coa2 protein may aid in stabilizing an early Cox1 intermediate containing the nuclear subunits Cox5a and Cox6.     

Effect of IUDs on uterine cyclic AMP rabbits.

IUDs were inserted into the uteri of 15 New Zealand White rabbits in order to observe the effect of IUDs on uterine cyclic AMP. Rabbits received either a plastic IUD, a plastic-copper IUD, or were untreated. After 1, 2, or 3 months, the uteri were removed and analyzed. In 3 animals, IUDs were removed after 2 months and uteri excised 1 month later. Cyclic AMP was measured in neutralized, acid-extracted supernatants by the radioimmunoassay procedure of Steiner et al. Cyclic AMP was significantly lower (p less than .04) in the uteri containing plastic or plastic-copper IUDs by 1 month and remained depressed for up to 3 months. Cyclic AMP levels returned to control values in animals in which the plastic IUD was removed at 2 months, but remained depressed after removal of the plastic-copper IUD. The delayed effect of Copper-containing IUDs on the return of fertility in the rabbit needs further study.     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.