Endogenous AP-1 levels necessary for oncogenic activity are higher than those sufficient to support normal growth

We investigated the role of endogenous AP-1 in human tumor cell lines by introducing SupJunD-1, a dominant-negative mutant of AP-1, using vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors. Single inoculation of six human tumor cell lines, originating from osteosarcomas, non-small cell lung carcinomas or cervical carcinomas, with recombinant SupJunD-1 virus at a high multiplicity of infection readily inhibited colony formation in soft agar. We detected no significant changes in expression levels of AP-1 components c-Jun or Fra-1, adhesion molecules CD44 or E-cadherin, or cell cycle regulator p53, which are encoded by genes previously reported to be under the control of AP-1 in some mouse or human cell lines. By varying the dosage of VSV-G-pseudotyped retrovirus, we were able to change the proviral copy number of supjunD-1 from 1 to approximately 10 and monitor suppression of endogenous AP-1 function as assessed by growth characteristics of the tumor cell lines, we found a SupJunD-1 dosage which significantly suppressed anchorage-independent growth without affecting the cellular growth in monolayer cultures at all. We conclude that endogenous AP-1 levels necessary for oncogenic activity are much higher than those sufficient to support normal growth.     

Soluble fumarate reductase isoenzymes from Saccharomyces cerevisiae are required for anaerobic growth

In Saccharomyces cerevisiae, the cytosolic and promitochondrial isoenzymes of fumarate reductase are encoded by the FRDS and OSM1 genes, respectively. The product of the OSM1 gene is reported to be required for growth in hypertonic medium. Simultaneous disruption of the FRDS and OSM1 genes resulted in the inability of the yeasts to grow anaerobically on glucose as a carbon source, and disruption of the OSM1 gene caused poor growth under anaerobic conditions. However, the disruption of both the FRDS and/or OSM1 genes had no effect on aerobic growth or growth under hypertonic conditions. These results suggest that the fumarate reductase isoenzymes in Saccharomyces cerevisiae are essential for anaerobic growth but not for growth under hypertonic conditions.     

Deoxyribonucleotide biosynthesis in yeast (Saccharomyces cerevisiae). A ribonucleotide reductase system of sufficient activity for DNA synthesis

Ribonucleotide reductase, the central enzyme of DNA precursor biosynthesis, has been isolated and characterized from baker's yeast. The enzyme activity, measured in extracts from three different, exponentially growing yeast strains, is high enough to meet the substrate requirement of DNA replication, in contrast to very low activities found in most other organisms. In thymidylate-permeable yeast cells ribonucleotide reductase activity is stimulated under both starvation and excess of intracellular dTMP. On the other hand growth of yeast in presence of 20 mM hydroxyurea did not increase enzyme activity. Yeast ribonucleotide reductase is composed of two non-identical subunits, inactive separately, of which one binds to immobilized dATP. The relative molecular mass of the holoenzyme is about 250 000. The enzyme reduces all four natural ribonucleoside diphosphates with comparable efficacy. GDP reduction requires dTTP as effector, ADP reduction is stimulated by dGTP, whereas pyrimidine nucleotide reduction is stimulated by any deoxyribonucleotide and ATP. Enzyme activity is independent of exogenous metal ions and is insensitive towards chelating agents. Hydroxyurea inactivates yeast ribonucleotide reductase in a slow reaction; half-inhibition (I50) is reached only at 2-6 mM hydroxyurea concentration. Up to 50% reactivation occurs spontaneously after removal of the inhibitor. In accord with previous attempts by others, extensive purification of the yeast enzyme has failed owing to its extreme instability in solution; the half-life of about 11 h could not be influenced by any protective measure. Taken together, yeast ribonucleotide reductase combines features known from Escherichia coli and mammalian enzymes with differing, individual properties.     

Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine.

By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation. Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase. The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation. These data can be explained by postulating the existence of two enzyme activities not previously reported in S. cerevisiae. The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-). The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).     

Regulation of S-adenosylmethionine levels in Saccharomyces cerevisiae

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine biosynthesis. Methionine can be activated by ATP to give rise to the universal methyl donor, S-adenosylmethionine (AdoMet). Previously, a chimeric MTHFR (Chimera-1) comprised of the yeast Met13p N-terminal catalytic domain and the Arabidopsis thaliana MTHFR (AtMTHFR-1) C-terminal regulatory domain was constructed (Roje, S., Chan, S. Y., Kaplan, F., Raymond, R. K., Horne, D. W., Appling, D. R., and Hanson, A. D. (2002) J. Biol. Chem. 277, 4056-4061). Engineered yeast (SCY4) expressing Chimera-1 accumulated more than 100-fold more AdoMet and 7-fold more methionine than the wild type. Surprisingly, SCY4 showed no appreciable growth defect. The ability of yeast to hyperaccumulate AdoMet was investigated by studying the intracellular compartmentation of AdoMet as well as the mode of hyperaccumulation. Previous studies have established that AdoMet is distributed between the cytosol and the vacuole. A strain expressing Chimera-1 and lacking either vacuoles (vps33 mutant) or vacuolar polyphosphate (vtc1 mutant) was not viable when grown under conditions that favored AdoMet hyperaccumulation. The hyperaccumulation of AdoMet was a robust phenomenon when these cells were grown in medium containing glycine and formate but did not occur when these supplements were replaced by serine. The basis of the nutrient-dependent AdoMet hyperaccumulation effect is discussed in relation to homocysteine biosynthesis and sulfur metabolism.     

Anaerobiosis revisited: growth of Saccharomyces cerevisiae under extremely low oxygen availability

Applied Microbiology and Biotechnology - The budding yeast Saccharomyces cerevisiae plays an important role in biotechnological applications, ranging from fuel ethanol to recombinant protein...     

Cell growth and population activity of Saccharomyces cerevisiae in two-stage continuous cultivation

Continuous culture in a cascade of vessels with the addition of supplemental nutrients to any stage permits adjustment of the physiological state of the culture in each stage to best achieve a desired performance goal. The yeast Saccharomyces cerevisiae in two-stage continuous cultivation was selected as a model system. With conditions in the first stage held constant- at a selected glucose concentration in the feed stream, dilution rate for the second stage was varied. Cell numbers, dry weight, glucose concentration, respiration coefficient, and titers of several enzymes were determined. The seed rate was defined as the ratio of glucose concentration in the feeds to stage 1 and to stage 2. At low seed rates, the calculated specific growth rate in the second stage was proportional to dilution rate. At higher seed rates, the specific growth rate based on dry weight behaved differently from that based on cell numbers, and the dependence on dilution rate was not linear.     

LSM1 over-expression in Saccharomyces cerevisiae depletes U6 snRNA levels

Lsm1 is a component of the Lsm1-7 complex involved in cytoplasmic mRNA degradation. Lsm1 is over-expressed in multiple tumor types, including over 80% of pancreatic tumors, and increased levels of Lsm1 protein have been shown to induce carcinogenic effects. Therefore, understanding the perturbations in cell process due to increased Lsm1 protein may help to identify possible therapeutics targeting tumors over-expressing Lsm1. Herein, we show that LSM1 over-expression in the yeast Saccharomyces cerevisiae inhibits growth primarily due to U6 snRNA depletion, thereby altering pre-mRNA splicing. The decrease in U6 snRNA levels causes yeast strains over-expressing Lsm1 to be hypersensitive to loss of other proteins required for production or function of the U6 snRNA, supporting a model wherein excess Lsm1 reduces the availability of the Lsm2-7 proteins, which also assemble with Lsm8 to form a complex that binds and stabilizes the U6 snRNA. Yeast strains over-expressing Lsm1 also display minor alterations in mRNA decay and demonstrate increased susceptibility to mutations inhibiting cytoplasmic deadenylation, a process required for both 5′-to-3′ and 3′-to-5′ pathways of exonucleolytic decay. These results suggest that inhibition of splicing and/or deadenylation may be effective therapies for Lsm1-over-expressing tumors.     

Phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol): sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) activity was characterized from the mitochondrial fraction of Saccharomyces cerevisiae. The pH optimum for the reaction was 7.0. Maximum activity was dependent on manganese (0.1 mM), magnesium (0.3 mM), or cobalt (1 mM) ions and the nonionic detergent Triton X-100 (1 mM). The apparent Km values for CDP-diacylglycerol and glycerol-3-phosphate were 33 and 27 microM, respectively. Optimal activity was at 30 degrees C with an energy of activation of 5.4 kcal/mol (1 cal = 4.1868 J). Phosphatidylglycerophosphate synthase activity was thermally labile above 40 degrees C. p-Chloromecuriphenylsulfonic acid, N-ethylmaleimide, and mercurous ions inhibited activity. Phosphatidylglycerophosphate synthase activity was partially solubilized from the mitochondrial fraction with 1% Triton X-100.     

Trehalase activity and its regulation during growth of Saccharomyces cerevisiae.

Trehalase activity decreased in 95% at the onset of the transition phase of growth of S. cerevisiae. The question which we raised was whether this phenomenon was due to proteolysis or to conversion of the enzyme to a less active form (dephosphorylation). Immunological methods allowed to identify the presence of the trehalase protein during cell growth. At the same stage of growth, an increase in the non-phosphorylated enzyme was detected "in vitro". Results utilizing mutant strains also indicated that regulation occurred by interconversion of forms. The same mechanism also seems to control trehalase activity in non proliferating conditions.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

Two MAPK-signaling pathways are required for mitophagy in Saccharomyces cerevisiae

Macroautophagy (hereafter referred to simply as autophagy) is a catabolic pathway that mediates the degradation of long-lived proteins and organelles in eukaryotic cells. The regulation of mitochondrial degradation through autophagy plays an essential role in the maintenance and quality control of this organelle. Compared with our understanding of the essential function of mitochondria in many aspects of cellular metabolism such as energy production and of the role of dysfunctional mitochondria in cell death, little is known regarding their degradation and especially how upstream signaling pathways control this process. Here, we report that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is required for the degradation of both mitochondria and peroxisomes (via pexophagy), whereas Hog1 functions specifically in mitophagy. Slt2 also affects the recruitment of mitochondria to the phagophore assembly site (PAS), a critical step in the packaging of cargo for selective degradation.