Influence of oxygen on the microsomal electron transport system in Saccharomyces cerevisiae

The influence of oxygen on the level of microsomal electron transport chain components has been studied during the growth of Saccharomyces cerevisiae. Enzyme activities and cytochrome content were assayed in microsomal fractions prepared from a protoplast lysate free from mitochondrial contamination. It was found that the cytochrome P-450 and cytochrome b5 content, to get her with the NADPH-cytochrome (P-450)-reductase and NADH-cytochrome (b5)-reductase activities, were increased in the cells as the pO2 of the medium was decreasing. At the same time an increase in the membrane surface of the endoplasmic reticulum can be observed.     

Effect of triazole fungicides on lipid metabolism of Saccharomyces cerevisiae

The triazole fungicide (Flusilazole) modified the sterol content of Saccharomyces cerevisiae. The plasma membrane fluidity was altered by the presence of methyl sterol which increased with the flusilazole concentration. On the other hand, the short free fatty acids (C6 to C14) and the unsaturated free fatty acids increased in the cells, while the short free fatty acids decreased in the medium.     

Effect of xylose incubation on the glucose transport system in Saccharomyces cerevisiae

Incubation of Saccharomyces cerevisiae with xylose and ethanol for 16 hours leads to a decrease of hexokinase (and glucokinase) activity in the cells. It does not alter the levels of polyphosphate, orthophosphate and ATP. The transport of the glucose derivative 2-deoxy-D-glucose, a sugar that can be phosphorylated, is inhibited after this treatment, whereas transport of 6-deoxy-D-glucose, which has a blocked phosphorylation site, is not inhibited. Even though, both deoxyglucoses use the same transport system. The decrease in initial velocity of 2-deoxy-D-glucose transport is most pronounced under anaerobic conditions. Incubation of the cells with antimycin A, a treatment which has a similar effect as anaerobiosis, shows, that the inhibition of the transport of 2-deoxy-D-glucose is presumably the result of an increase in the Km of the carrier transport. Transport of glucose is probably regulated by kinase enzymes.     

Dual system for potassium transport in Saccharomyces cerevisiae.

In a newly formulated growth medium lacking Na+ and NH4+, Saccharomyces cerevisiae grew maximally at 5 microM K+. Cells grown under these conditions transported K+ with an apparent Km of 24 microM, whereas cells grown in customary high-K+ medium had a significantly higher Km (2 mM K+). The two types of transport also differed in carbonyl cyanide-m-chlorophenyl hydrazone sensitivity, response to ATP depletion, and temperature dependence. The results can be accounted for either by two transport systems or by one system operating in two different ways.     

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.     

Effect of ethanol and other alkanols on the maltose transport system of Saccharomyces cerevisiae

Summary Maltose transport in S. cerevisiae was inhibited by ethanol and other alkanols in a non-competitive way. The Michaelis constant, K_m, for the sugar, with or without alkanols was 5.9 mM, whereas the maximum trans port capacity, V_max, decreased exponentially with alkanols concentration. The inhibitory capacity was positively correlated with the lipid solubility of the alkanols, indicating that inhibition is due to an alteration of the lipid environment of the maltose transport system in the plasma membrane.     

Effect of tunicamycin on thiamine transport in Saccharomyces cerevisiae

The activity of thiamine transport in Saccharomyces cerevisiae was decreased by the treatment with tunicamycin without affecting the growth of yeast cells. Although the total activity of a soluble thiamine-binding protein in yeast periplasm, which is known to be a glycoprotein, was decreased by tunicamycin treatment, the activity of thiamine uptake by yeast protoplasts was inhibited as much as by whole cells. Furthermore, tunicamycin decreased the activity of the membrane-bound thiamine-binding protein in a dose dependent way and in parallel with the thiamine transport activity. These findings suggested that the membrane-bound thiamine-binding protein is a glycoprotein which plays a functional role in thiamine transport in S. cerevisiae.     

Cloning and disruption of Ustilago maydis genes.

We have demonstrated that genes from Ustilago maydis can be cloned by direct complementation of mutants through the use of genomic libraries made in a high-frequency transformation vector. We isolated a gene involved in amino acid biosynthesis as an illustrative example and showed that integrative and one-step disruption methods can be used to create null mutations in the chromosomal copy of the gene by homologous recombination. The results of this investigation make it clear that one-step gene disruption will be of general utility in investigations of U. maydis, since simple, precise replacement of the sequence under study was readily achieved.     

The kinetics of the active and de-energized transport of O-methyl glucose in Ustilago maydis.

The kinetics of the uptake and efflux of 3-O-methyl-glucose in sporidia of Ustilago maydis were measured, both in active cells and in cells whose metabolic activity had been inhibited by azide and iodoacetate. The de-energized transport system proved to be carrier mediated with apparent affinity constants 13 +/- 2 mM outside (Ko) and 18 +/- 2 mM inside (K1). The apparent maximum rate constants for the same system were 0.66 +/- 0.05 mmol/1 cell water per min for uptake (V+) and 0.53 +/- 0.04 mmol/l cell water per min for efflux (V-). For the active system K0 = 0.08 +/- 0.01, K1 greater than 40, V+ = 9.7 +/- 0.5 and V- = 1.1 +/- 0.9 (in equivalent units). These results are discussed in the context of the carrier mechanism as proposed by Regen and Morgan (Regen, D.M. and Morgan, H.E. (1964) Biochim. Biophys. Acta 79, 151--166). The antifungal compound carboxin had no effect on de-energized transport but was shown to decrease both K0 And V+ in the active system. Phloretin and phlorizin were also found to be without effect on de-energized cells but the former enhanced while the latter inhibited active uptake.     

Phosphate transport and sensing in Saccharomyces cerevisiae.

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Delta cells; however, the pho84Delta strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Delta cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Delta strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters.     

Characterization of the biotin transport system in Saccharomyces cerevisiae

The characteristics of the biotin transport mechanism of Saccharomyces cerevisiae were investigated in nonproliferating cells. Microbiological and radioisotope assays were employed to measure biotin uptake. The vitamin existed intracellularly in both free and bound forms. Free biotin was extracted by boiling water. Chromatography of the free extract showed it to consist entirely of d-biotin. Cellular bound biotin was released by treating cells with 6 n H(2)SO(4). The rate of biotin uptake was linear with time for 10 min, reaching a maximum at about 20 min followed by a gradual loss of accumulated free vitamin from the cells. Biotin was not degraded or converted to vitamers during uptake. Transport was temperature- and pH-dependent, optimum conditions for uptake being 30 C and pH 4.0. Glucose markedly stimulated biotin transport. In its presence, large intracellular free-biotin concentration gradients were established. Iodoacetate inhibited the glucose stimulation of biotin uptake. The rate of vitamin transport increased in a linear fashion with increasing cell mass. The transport system was saturated with increasing concentrations of the vitamin. The apparent K(m) for uptake was 3.23 x 10(-7)m. Uptake of radioactive biotin was inhibited by unlabeled biotin and a number of analogues including homobiotin, desthiobiotin, oxybiotin, norbiotin, and biotin sulfone. Proline, hydroxyproline, and 7,8-diaminopelargonic acid did not inhibit uptake. Unlabeled biotin and desthiobiotin exchanged with accumulated intracellular (14)C-biotin, whereas hydroxyproline did not.     

Inactivation of the galactose transport system in Saccharomyces cerevisiae

The galactose transport system of Saccharomyces cerevisiae consists of one component which shows a Km value of approx. 4mM in growing cells. A rapid and irreversible inactivation of this transport is detected on impairment of protein synthesis. This inactivation shows the following characteristics: (i) it is due to changes in the Km and Vmax of the transport system; (ii) it follows first-order kinetics; (iii) it is an energy-dependent process and is stimulated by the presence of an exogenous carbon source; (iv) fermentable sub-dependent process and is stimulated by the presence of an exogenous carbon source; (iv) fermentable substrates stimulate inactivation more efficiently than non-fermentable substrates.     

Inhibition of biosynthesis of Saccharomyces cerevisiae sugar transport system by tunicamycin.

Tunicamycin apparently inhibited the biosynthesis of glucose, galactose, and maltose transport systems in Saccharomyces cerevisiae. Under the conditions used, the antibiotic also blocked the biosynthesis of invertase, a well-known yeast glycoprotein, as well as the glycosylation of a marker mannoprotein of the yeast cell wall. However, the antibiotic did not affect certain proteins which did not contain carbohydrate. It seems, therefore, that these sugar carriers are glycoproteins.     

Methylamine and ammonia transport in Saccharomyces cerevisiae.

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia.