Involvement of L-carnitine in cellular metabolism: beyond Acyl-CoA transport.

Carnitine is well-known for its role in the transport of fatty acids to the mitochondrial matrix, where beta-oxidation takes place. This work describes novel functions for this compound and novel data on its pharmacokinetics.

Effect of potassium on Saccharomyces cerevisiae resistance to fluconazole.

Susceptibility of strain S288c of Saccharomyces cerevisiae to fluconazole was assayed in the presence and absence of KCl. Addition of 150 mM KCl renders the strain more sensitive to the antifungal agent. The effect is caused by the K(+) ion rather than the anion or the osmolarity of the medium. The increase in sensitivity does not modify the values of intracellular and extracellular pH established in the presence of KCl.

[Relationship between rhodamine 6G accumulation and fluconazole resistance in Saccharomyces cerevisiae S288c]. / Relación entre acumulación de rodamina 6G y resistencia a fluconazol en Saccharomyces cerevisiae S288c.

Severe mycotic infections are a source of concern in immunocompromised patients or in those who receive chemotherapy for hematological malignant diseases. One of the causes is referred to the appearance of antimycotic resistant microorganisms. Fluconazole is one of the antimycotic used for invasive mycoses treatment. Therefore it is necessary to evaluate the factors that originate this resistance. In the present report the yeast Saccharomyces cerevisiae S288c was used as a model system. In resistant strains the accumulation of the lipophilic cation Rhodamine 6G, L-leucine uptake and growth inhibition by crystal violet dye were determined. The results presented herein demonstrate the correlation between the membrane potential and the resistance to fluconazole presented by S. cerevisiae strain S288c.

Relación entre acumulación de rodamina 6G y resistencia a fluconazol en Saccharomyces cerevisiae S288c / Relationship between rhodamine 6G accumulation and fluconazole resistance in Saccharomyces cerevisiae S288c

Severe mycotic infections are a source of concern in immunocompromised patients or in those who receive chemotherapy for hematological malignant diseases. One of the causes is referred to the appearance of antimycotic resistant microorganisms. Fluconazole is one of the antimycotic used for invasive mycoses treatment. Therefore it is necessary to evaluate the factors that originate this resistance. In the present report the yeast Saccharomyces cerevisiae S288c was used as a model system. In resistant strains the accumulation of the lipophilic cation Rhodamine 6G, L-leucine uptake and growth inhibition by crystal violet dye were determined. The results presented herein demonstrate the correlation between the membrane potential and the resistance to fluconazole presented by S. cerevisiae strain S288c.

Relación entre acumulación de rodamina 6G y resistencia a fluconazol en Saccharomyces cerevisiae S288c / Relationship between rhodamine 6G accumulation and fluconazole resistance in Saccharomyces cerevisiae S288c

Severe mycotic infections are a source of concern in immunocompromised patients or in those who receive chemotherapy for hematological malignant diseases. One of the causes is referred to the appearance of antimycotic resistant microorganisms. Fluconazole is one of the antimycotic used for invasive mycoses treatment. Therefore it is necessary to evaluate the factors that originate this resistance. In the present report the yeast Saccharomyces cerevisiae S288c was used as a model system. In resistant strains the accumulation of the lipophilic cation Rhodamine 6G, L-leucine uptake and growth inhibition by crystal violet dye were determined. The results presented herein demonstrate the correlation between the membrane potential and the resistance to fluconazole presented by S. cerevisiae strain S288c.(AU)

Relación entre acumulación de rodamina 6G y resistencia a fluconazol en Saccharomyces cerevisiae S288c. / [Relationship between rhodamine 6G accumulation and fluconazole resistance in Saccharomyces cerevisiae S288c]

Severe mycotic infections are a source of concern in immunocompromised patients or in those who receive chemotherapy for hematological malignant diseases. One of the causes is referred to the appearance of antimycotic resistant microorganisms. Fluconazole is one of the antimycotic used for invasive mycoses treatment. Therefore it is necessary to evaluate the factors that originate this resistance. In the present report the yeast Saccharomyces cerevisiae S288c was used as a model system. In resistant strains the accumulation of the lipophilic cation Rhodamine 6G, L-leucine uptake and growth inhibition by crystal violet dye were determined. The results presented herein demonstrate the correlation between the membrane potential and the resistance to fluconazole presented by S. cerevisiae strain S288c.

The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport.

Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize L-leucine as sole nitrogen source and very low levels of [14C]L-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]L-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic.

L-proline as a nitrogen source increases the susceptibility of Saccharomyces cerevisiae S288c to fluconazole.

Fluconazole inhibition of Saccharomyces cerevisiae S288c growth was evaluated in media containing ammonia, L-proline or L-leucine as a nitrogen source. Growth inhibition by fluconazole was maximum when L-proline was used as a nitrogen source, while rhodamine 6G accumulation and fluconazole resistance were the highest when ammonia was the sole nitrogen source.

RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae.

The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.

Isolation of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae deficient in both high- and low-affinity L-leucine transport.

A yeast mutant defective in permeases S1 and S2 which transport L-leucine was isolated from a parental strain already deficient in the general amino acid permease, GAP1. The mutant was selected as a spontaneous, trifluoroleucine-resistant (TFLR) strain. Full resistance depended upon the presence of two unlinked mutant genes designated let1 and let2. The let1 mutation completely inactivates the high-affinity leucine transport system defined kinetically as S1. Although the let2 mutation caused a marked decrease in the Jmax of the low-affinity transport system, S2, residual leucine transport in the let1 let2 gap1 mutant had the same KT as in the LET1 LET2 gap1 parent. The mutant exhibited a marked decrease in growth on minimal medium containing leucine, isoleucine or valine as a sole nitrogen source. Moreover, assimilation of methionine, phenylalanine, serine and threonine was decreased, whereas basic and acidic amino acids supported normal growth. This indicates that at least one of the leucine permeases has a fairly broad, but still limited, specificity. Reversion of the gap1 gene restored leucine transport. The revertant was sensitive to TFL when grown on proline but resistant when NH4+ was the nitrogen source. The previously published mutations (shr3, aat1, lup1 or raa) would not be related to either LET1 or LET2.

Why Saccharomyces cerevisiae can oxidize but not decarboxylate external pyruvate.

Protoplasts of the yeast Saccharomyces cerevisiae oxidized externally added pyruvate by pyruvate oxidase system but were not able to decarboxylate it anaerobically by pyruvate decarboxylase at pH 6.4 in isotonic solutions. The decarboxylation set in hypotonic solutions in which the integrity of the plasma membrane was being impaired. Yeast cells incubated with [1-14C]pyruvate accumulated radioactivity under conditions allowing oxidation of pyruvate, but virtually no pyruvate was taken up when the oxidation had been arrested by inhibition or mutation. In view of a large difference between Km for pyruvate of pyruvate decarboxylase (30 mM) and of pyruvate oxidase (0.16 mM), the results may be accounted for by the assumption that transport of pyruvate across the yeast plasma membrane is trans-inhibited by relatively high concentrations of intracellular pyruvate. This arrangement would allow utilization of external pyruvate by the cell energy-transforming machinery and, at the same time, prevent its wastage by futile decarboxylation.

L-leucine transport systems in Saccharomyces cerevisiae participation of GAP1, S1 and S2 transport systems.

L-leucine uptake in Saccharomyces cerevisiae is mediated by three different transport systems, S1, S2 and GAP1. Their activities are dependent on the nitrogen source of the culture media. Wild type cells grown in L-proline exhibit a single transport system with high affinity and high Vmax that is partially inhibited by L-citrulline. A gap1 mutant shows two transport systems with Km and Vmax values similar to those previously described as S1 and S2, this transport activity is not inhibited by D-leucine, D-isoleucine or D-valine. Two systems can be also determined in wild type cells grown in rich medium containing a mixed nitrogen source where decreased GAP1 function is observed. In either wild type or gap1 cells grown in medium containing ammonium ions as sole nitrogen source, L-leucine uptake kinetics shows two systems with lower Vmax and similar Km values to those of the S1 and S2 systems. These results show that in S. cerevisiae GAP1, S1 and S2 participate in L-leucine entrance in cells grown in a poor nitrogen source, and that S1 and S2 are two ammonia-sensitive permeases that mediate the uptake in cells grown in a rich nitrogen source.

Inhibitory action of palmitic acid on the growth of Saccharomyces cerevisiae.

High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.

Study on fatty acid binding by proteins in yeast. Dissimilar results in Saccharomyces cerevisiae and Yarrowia lipolytica.

1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.

[Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae. Repression and inhibition of transport systems]. / Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae. Represión e inhibición de los sistemas transportadores.

L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.

Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae. Represión e inhibición de los sistemas transportadores / [Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae. Repression and inhibition of transport systems]

L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.

Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae. Represión e inhibición de los sistemas transportadores. / [Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae. Repression and inhibition of transport systems]

L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.