Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Accumulation and intracellular compartmentation of lithium ions in Saccharomyces cerevisiae

Abstract Accumulation of Li⁺ in Saccharomyces cerevisiae X2180-1B occured via an apparent stoichiometric relationship of 1: 1 (K⁺/Li⁺) when S. cerevisiae was incubated in the presence of 5 and 10 mM LiCl for 3 h. Other cellular cations (Mg²⁺, Ca²⁺ and Na⁺) did not vary on Li⁺ accumulation, although lithium chemistry dictates a degree of similarity to Group I and II metal cations. Compartmentation of Li⁺ was mainly in the vacuole which accounted for 85% of the Li⁺ accumulated after a 6-h incubation period. The remainder was located in the cytosol with negligible amounts being bound to cell fragments including the cell wall. Transmission electron microscopy of Li⁺-loaded cells revealed enlarged vacuoles compared with control cells. This asymmetric cellular distribution may therefore enhance tolerance of S. cerevisiae to Li⁺ and ensure that essential metabolic processes in the cytosol are not disrupted.     

Intermediary Metabolite Concentrations in Xylulose- and Glucose-Fermenting Saccharomyces cerevisiae Cells

Glucose and xylulose fermentation and product formation by Saccharomyces cerevisiae were compared in batch culture under anaerobic conditions. In both cases the main product was ethanol, with glycerol, xylitol, and arabitol produced as by-products. During glucose and xylulose fermentation, 0.74 and 0.37 g of cell mass liter⁻¹, respectively, were formed. In glucose-fermenting cells, the carbon balance could be closed, whereas in xylulose-fermenting cells, about 25% of the consumed sugar carbon could not be accounted for. The rate of sugar consumption was 3.94 mmol g of initial biomass⁻¹ h⁻¹ for glucose and 0.39 mmol g of initial biomass⁻¹ h⁻¹ for xylulose. Concentrations of the intermediary metabolites fructose-1,6-diphosphate (FDP), pyruvate (PYR), sedoheptulose 7-phosphate (S7P), erytrose 4-phosphate, citrate (CIT), fumarate, and malate were compared for both types of cells. Levels of FDP, PYR, and CIT were lower, and levels of S7P were higher in xylulose-fermenting cells. After normalization to the carbon consumption rate, the levels of FDP were approximately the same, whereas there was a significant accumulation of S7P, PYR, CIT, and malate, especially of S7P, in xylulose-fermenting cells compared with in glucose-fermenting cells. In the presence of 15 μM iodoacetate, an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), FDP levels increased and S7P levels decreased in xylulose-assimilating cells compared with in the absence of the inhibitor, whereas fermentation was slightly slowed down. The specific activity of transaldolase (EC 2.2.1.2), the pentose phosphate pathway enzyme reacting with S7P and glyceraldehyde-3-phosphate, was essentially the same for both glucose- and xylulose-fermenting cells. It was, however, several orders of magnitude lower than that reported for a Torula yeast and Candida utilis. The presence of iodoacetate did not influence the activity of transaldolase in xylulose-fermenting cells. The results are discussed in terms of a competition between the pentose phosphate pathway and glycolysis for the common metabolite, glyceraldehyde-3-phosphate, which would explain the low rates of xylulose assimilation and ethanol production from xylulose by S. cerevisiae.     

Subcellular compartmentation in control of converging pathways for proline and arginine metabolism in Saccharomyces cerevisiae.

Enzymes of proline biosynthesis and proline degradation which act on the same compound, delta 1-pyrroline-5-carboxylate, are physically separated in yeast cells. The enzyme responsible for the final step in proline biosynthesis, pyrroline-5-carboxylate reductase, converts pyrroline-5-carboxylate to proline and is located in the cytoplasm. The last enzyme in the proline degradative pathway, pyrroline-5-carboxylate dehydrogenase, converts pyrroline-5-carboxylate to glutamate and is found in the particulate fraction of the cell, presumably in the mitochondrion. By subcellular compartmentation, yeast cells avoid futile cycling between proline and pyrroline-5-carboxylate.     

Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae

The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.     

Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae

Three enolase isoenzymes can be distinguished after electrophoresis of yeast crude extracts. After adding glucose to derepressed cells, there was a coordinated increase in the activity of enolase I and decrease in enolase II activity. Enolase I was found to be repressed and enolase II simultaneously induced by glucose. The third enolase activity remained unchanged and was identified as that of a hybrid enzyme. Enolase catalyses the first common step of glycolysis and gluconeogenesis. Gluconeogenic enolase I shows substrate inhibition for 2-phosphoglycerate (glycolytic substrate) and glycolytic enolase II is substrate-inhibited by phosphoenolpyruvate (gluconeogenic substrate). The gluconeogenic reaction was inhibited up to 45% by physiological concentrations of fructose 1,6-bisphosphate. To test for cytological compartmentation, a method was developed for isolating microsomes. Effective enrichment of rough and smooth endoplasmic reticulum was demonstrated by electron microscopy. No evidence was obtained for any compartmentation of either enolases or other glycolytic enzymes.     

Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae.

A mutation in Saccharomyces cerevisiae, designated mia, is responsible for the production of isoaccepting tRNA molecules with reduced extents of nucleoside modifications. The mia isoacceptors of tRNAPhe and one of the mutant isoacceptors of tRNATyr were highly purified for nucleoside composition analyses. The data indicate that the mutant isoacceptors are lacking some of the dihydrouridine moieties. This is consistent with our previous hypothesis that the mutant isoacceptors were accumulated due to a defect in a modification process [Lo, R.Y.C. and Bell, J.B. (1981) Current Genetics 3, 73-82). Data from in vitro poly-U translation experiments also support the previous results, suggesting in vivo biological activity of these mutant tRNAs.     

Sexual agglutination in Saccharomyces cerevisiae.

Treatment of either mating type of Saccharomyces cerevisiae with the appropriate sex pheromone increased cell-cell binding in a modified cocentrifugation assay. Constitutive agglutination of haploids was qualitatively similar to pheromone-induced agglutination. Regardless of exposure to pheromone, agglutinable combinations of cells exhibited maximal binding across similar ranges of ionic strength, pH, and temperature. Binding of all combinations was inhibited by 8 M urea, 1 M pyridine, or 0.05% sodium dodecyl sulfate. From alpha-cells we solubilized and partially purified an inhibitor of a-cell agglutinability. This inhibitor reversibly masked all a-cell adhesion sites and inactivated pheromone-treated and control cells with similar kinetics. The inhibitor behaved as a homogeneous species in heat inactivation experiments. Based on these results, we proposed a model for pheromone effects on agglutination in S. cerevisiae.     

Allantoin transport in Saccharomyces cerevisiae.

Allantoin uptake in both growing and resting cultures of Saccharomyces cerevisiae occurs by a low-Km (ca. 15 micrometer) transport system that uses energy that is likely generated in the cytoplasm. This conclusion was based on the observation that transport did not occur in the absence of glucose or the presence of dinitrophenol, carbonyl cyanide-m-chloro-phenyl hydrazine, fluoride, or arsenate ions. Normal uptake was observed, however, in the presence of cyanide. The rate of accumulation was maximal at pH 5.2. In contrast to the urea transport system, allantoin uptake appeared to be unidirectional. Preloaded, radioactive allantoin was not lost from cells suspended in allantoin-free buffer and did not exchange with exogenously added, nonradioactive allantoin. Treatment of preloaded cells with nystatin, however, released the accumulated radioactivity. Allantoin accumulated within cells was isolated and shown to be chemically unaltered.     

Peptidase activities in Saccharomyces cerevisiae.

At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives.     

Purine metabolism in Saccharomyces cerevisiae.

The synthesis, interconversion, and catabolism of purine bases, ribonucleosides, and ribonucleotides in wild-type Saccharomyces cerevisiae were studied by measuring the conversion of radioactive adenine, hypoxanthine, guanine, and glycine into acid-soluble purine bases, ribonucleosides, and ribonucleotides, and into nucleic acid adenine and guanine. The pathway(s) by which adenine is converted to inosinate is (are) uncertain. Guanine is extensively deaminated to xanthine. In addition, some guanine is converted to inosinate and adenine nucleotides. Inosinate formed either from hypoxanthine or de novo is readily converted to adenine and guanine nucleotides.     

Postreplication repair in Saccharomyces cerevisiae.

Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light (2 to 4 J/m2). Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were "chased," the interruptions were no longer detected. Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, we concluded that postreplication repair in excision-defective mutants (or leaky mutants) does not involve molecular recombination. Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.     

Sterol methylation in Saccharomyces cerevisiae.

Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5.     

Choline transport in Saccharomyces cerevisiae.

Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and various amino acids examined had no effect. Choline transport required metabolic energy; removal of glucose resulted in a great loss of transport activity, and the remaining activity was abolished by 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, arsenate, and cyanide. External Na+ was not required, and the transport was not effected by ionophores, valinomycin, and gramicidin D. These results indicate that S. cerevisiae possess an active choline transport system mediated by a specific carrier. This view is further supported by the isolation and characterization of a choline transport mutant. The choline transport activity in this mutant was very low, whereas the transport of L-leucine, L-methionine, D-glucose, and myo-inositol was normal. Together with the choline transport mutant, mutants defective in choline kinase were also isolated.     

Allantoate transport in Saccharomyces cerevisiae.

Allantoate uptake appears to be mediated by an energy-dependent active transport system with an apparent Michaelis constant of about 50 microM. Cells were able to accumulate allantoate to greater than 3,000 times the extracellular concentration. The rate of accumulation was maximum at pH 5.7 to 5.8. The energy source for allantoate uptake is probably different from that for uptake of the other allantoin pathway intermediates. The latter systems are inhibited by arsenate, fluoride, dinitrophenol, and carboxyl cyanide-m-chlorophenyl hydrazone, whereas allantoate accumulation was sensitive to only dinitrophenol and carboxyl cyanide-m-chlorophenyl hydrazone. Efflux of preloaded allanotate did not occur at detectable levels. However, exchange of intra- and extracellular allantoate was found to occur very slowly. The latter two characteristics are shared with the allantoin uptake system and may result from the sequestering of intracellular allantoate within the cell vacuole. During the course of these studies, we found that, contrary to earlier reports, the reaction catalyzed by allantoinase is freely reversible.     

Myo-inositol transport in Saccharomyces cerevisiae.

myo-Inositol uptake in Saccharomyces cerevisiae was dependent on temperature, time, and substrate concentration. The transport obeyed saturation kinetics with an apparent Km for myo-inositol of 0.1 mM, myo-Inositol analogs, such as scyllo-inositol, 2-inosose, mannitol, and 1,2-cyclohexanediol, had no effect on myo-inositol uptake, myo-Inositol uptake required metabolic energy. Removal of D-glucose resulted in a loss of activity, and azide and cyanide ions were inhibitory. In the presence of D-glucose, myo-inositol was accumulated in the cells against a concentration gradient. A myo-inositol transport mutant was isolated from UV-mutagenized S. cerevisiae cells using the replica-printing technique. The defect in myo-inositol uptake was due to a single nuclear gene mutation. The activities of L-serine and D-glucose transport were not affected by the mutation. Thus it was shown that S. cerevisiae grown under the present culture conditions possessed a single and specific myo-inositol transport system. myo-Inositol transport activity was reduced by the addition of myo-inositol to the culture medium. The activity was reversibly restored by the removal of myo-inositol from the medium. This restoration of activity was completely abolished by cycloheximide.     

Proline transport in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is capable of utilizing proline as the sole source of nitrogen. Mutants of S. cerevisiae with defective proline transport were isolated by selecting for resistance to either of the toxic proline analogs L-azetidine-2-carboxylate or 3,4-dehydro-DL-proline. Strains carrying the put4 mutation are defective in the high-affinity proline transport system. These mutants could still grow when given high concentrations of proline, due to the operation of low-affinity systems whose existence as confirmed by kinetic studies. Both systems were repressed by ammonium ions, and either was induce by proline. Low-affinity transport was inhibited by histidine, so put4 mutants were unable to grow on a medium containing high concentrations of proline to which histidine has been added.     

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.     

Cystathionine accumulation in Saccharomyces cerevisiae.

A cysteine-dependent strain of Saccharomyces cerevisiae and its prototrophic revertants accumulated cystathionine in cells. The cystathionine accumulation was caused by a single mutation having a high incidence of gene conversion. The mutation was designated cys3 and was shown to cause loss of gamma-cystathionase activity. Cysteine dependence of the initial strain was determined by two linked and interacting mutations, cys3 and cys1 . Since cys1 mutations cause a loss of serine acetyltransferase activity, our observation led to the conclusion that S. cerevisiae synthesizes cysteine by sulfhydrylation of serine with hydrogen sulfide and by cleavage of cystathionine which is synthesized from serine and homocysteine.