Physiology of lysine permeases in Saccharomycopsis lipolytica.

Two active lysine transport systems were detected in Saccharomycopsis lipolytica. No excretion of lysine out of the cells could be obtained, even by chasing with L-lysine or by poisoning with sodium azide. The kinetic properties of one of the permeases, the high-affinity lysine permease, were studied in detail. Its Km was 1.91 +/- 0.23 X 10(-5) M. It proved highly specific, the only potent competitive inhibitors being (i) arginine and its analogs L-canavanine and L-ornithine, and (ii) the lysine analogs L-5 aminoethylcysteine and L-4,5-transdehydrolysine. It is suggested that the high-affinity lysine permease is common to L-lysine, L-ornithine, and L-arginine. The other amino acids tested behaved as noncompetitive inhibitors. The variation of uptake during a growth cycle was studied on ammonia-rich, ammonia-poor, and ammonia-free media. In each case, the uptake exhibited a peak in the early exponential growth phase. No new permease activity was detected during the lag phase or the stationary phase. Ammonia ions competitively inhibited the uptake and also decreased the Vmax value.     

Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests.     

Preliminary genetic studies on a citric acid producing strain of Saccharomycopsis lipolytica

The genetics of a strain of Saccharomycopsis lipolytica which accumulates citric acid have been studied. The results show that a mating system exists in the organism although no diploid was isolated.     

Regulation of the central metabolism in relation to citric acid production in Saccharomycopsis lipolytica

The mechanism of the massive extracellular production of citric and isocitric acids by Saccharomycopsis lipolytica grown on n-paraffins has been studied. When growth stops, because of nitrogen limitation, the intracellular concentration of ATP sharply rises whereas that of AMP and ADP decreases to a low level. At the same time production of acids begins. The activity of the NAD-dependent isocitrate dehydrogenase which requires AMP for activity becomes very low and prevents the oxidative function of the citric acid cycle whereas isocitrate lyase is not inhibited. As citrate synthase inhibition by ATP appears to be insufficient to stop n-paraffin degradation, citric and isocitric acids accumulation can take place. Massive excretion of these acids, however, probably still involves other physiological changes brought about by nitrogen limitation, possibly some permeabilization of the cell to these acids.This work is a part of a Doctorat de Spécialité Thesis submitted by R. Marchal to the University of Nancy (1975)     

Amino acids control ammonia pulses in yeast colonies

Individual yeast colonies produce pulses of volatile ammonia separated by phases of medium acidification. Colonies of Saccharomyces cerevisiae mutant defective in the general amino acid permease, Gap1p, exhibit decreased ammonia production. Mutations in the S. cerevisiae amino acid sensor SPS completely abolish the colony ammonia pulses. In contrast, the ammonia pulse production is independent of external concentrations of ammonium and of its uptake by the ammonium permeases Mep1p, Mep2p, and Mep3p. It is concluded that in S. cerevisiae colonies, the extracellular amino acids, but not the extracellular ammonium, serve as a source for volatile ammonia production. These phenomena are not restricted to S. cerevisiae, since we observe that extracellular levels of 8 out of the 20 tested amino acids are necessary for ammonia pulses produced by Candida mogii colonies.     

Reversion by Fe(III) of the inhibition by hydroxamic acids of the cyanide-Insensitive respiration in the yeast Saccharomycopsis lipolytica

The specific inhibitory effect of benzhydroxamic acid on the cyanide-insensitive respiration could be reversed in whole cells of the yeast Saccharomycopsis lipolytica, by addition of Fe(III), in a way suggesting a competition between the added iron and an enzyme-bound metallic ion, both central atoms for the ligand benzhydroxamic acid. The possibility that added metal ions modify the penetration of BHAM into the cells was ruled out. Co(II), Cu(II) and Al(III) could substitute for Fe(III). A linear relation between the concentration in added Fe(III) and the reversed respiration rate was observed. At a given cell concentration. the reversion by added Fe(III) of the inhibitory effect of benzhydroxamic acid on the alternative respiration appeared more related to the degree of inhibition rather than to the concentration in added inhibitor. Increasing cell concentrations required increasing amounts of Fe(III) to reach the same level of reversion. No reversal occurred at concentrations in added Fe(III) lower than 0.1 mM, whatever the benzhydroxamic concentration, the cell concentration or the yeast batch.     

Genetic control of amino acid transport in sheep erythrocytes

An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr ^H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr ^h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr ^H gene behaves as if codominant to Tr ^h. Erythrocytes from sheep homozygous for the Tr ^H gene exhibit rapid saturable l-alanine influx (apparent K _m ,21.6mm; V _max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr ^h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V _max (apparent K _m, 19.1mm; V _max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr ^H, Tr^H sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant.     

Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow

Abstract Threonine and lysine are two of the economically most important essential amino acids. They are produced industrially by species of the genera Corynebacterium and Brevibacterium . The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms. This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids. Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine. Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis).     

A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter,LeuT

Abstract

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.     

Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source

Lipid particles (LP) of all types of cells are a depot of neutral lipids. The present investigation deals with the isolation of LP from the yeast Yarrowia lipolytica and the characterization of their lipid and protein composition. Properties of LP varied depending on the carbon source. LP from glucose-grown cells revealed a mean diameter of 650 nm with a hydrophobic core mainly formed of triacylglycerols (TAG) and a minor amount of steryl esters (SE). Oleic acid was the major fatty acid species esterified in LP. When cells were grown on oleic acid, LP size increased 3.8-fold, the particles exhibited a significantly lower ratio of TAG to SE, and the relative amount of oleic acid in LP lipids increased compared to cells grown on glucose. Analysis of LP proteins revealed an increasing number of polypeptides when cells were shifted from glucose- to oleic acid-containing medium. Twenty-one major LP proteins were identified under both growth conditions, and additional nine polypeptides were specific for growth on oleic acid. Identification of these proteins by MS and comparison of the deduced ORFs to those from Saccharomyces cerevisiae revealed that most proteins of Y. lipolytica LP are involved in lipid metabolism. LP proteins specific for growth on oleic acid are also enzymes involved in lipid metabolism, but some of them are also components of the intracellular traffic machinery. Thus, proteom analysis of LP proteins suggests involvement of this compartment in different cell biological processes.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

Post genomic analysis of permeases from the amino acid/auxin family in protozoan parasites

The "amino acid/auxin permeases" is probably the most represented family of transporters in the Trypanosoma cruzi genome. Using a high-throughput searching routine and preliminary data from the T. cruzi genome project, more than 15,000 sequences were iteratively assembled into contigs, and 60 open reading frames corresponding to different putative amino acid transporters, clustered in 12 groups, were detected and characterized in silico. T. cruzi genomic organization of such sequences showed that these putative amino acid transporter genes are in an unusually large number and arranged in repeat clusters comprising about 0.2% of the genome. These data suggest that the family has evolved following tandem duplication events and constitutes a novel family of variable proteins in protozoan organisms. The mRNA expression of the predicted genes was demonstrated in infective and non-infective parasite forms. Orthologous sequences were also identified in other unicellular parasites such as Leishmania spp., Plasmodium spp., and Trypanosoma brucei.     

Energetics of membrane transport in protoplasts

Examples are given to illustrate the recent use of isolated protoplasts in the study of membrane transport with the emphasis on the energetics of solute transport. A model is also presented for the mechanism of active solute transport at the plasmalemma.     

Amiodarone inhibits multiple drug resistance in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Amiodarone is a widely used antiarrhythmic drug. There is also evidence that amiodarone decreases multidrug resistance in human cell lines. In this paper, we have shown that amiodarone has similar effect on yeast, Saccharomyces cerevisiae, decreasing multiple drug resistance. Amiodarone stimulates the accumulation of ethidium bromide by inhibiting its efflux from the cells. The effect of amiodarone is much stronger on wild-type cells compared to the mutant with inactivated ABC-transporters. Interestingly, the action of amiodarone is additive with the one of chloroquine, a known inhibitor of ABC-transporters. We speculate that these findings could help in the development of antifungal drug mixes.     

Secondary Transport of Amino Acids in Prokaryotes

Amino acid transport is a ubiquitous phenomenon and serves a variety of functions in prokaryotes, including supply of carbon and nitrogen for catabolic and anabolic processes, pH homeostasis, osmoprotection, virulence, detoxification, signal transduction and generation of electrochemical ion gradients. Many of the participating proteins have eukaryotic relatives and are successfully used as model systems for exploration of transporter structure and function. Distribution, physiological roles, functional properties, and structure-function relationships of prokaryotic α-amino acid transporters are discussed.     

Polarized nature of taurine transport in LLC-PK1 and MDCK cells: Further characterization of divergent transport models

Summary Taurine transport was measured in cultured epithelial cells-LLC-PK1 and MDCK-grown on permeable membrane supports. Taurine transport by LLC-PK1 cells was greater on the apical surface compared to the basolateral surface. MDCK cells exhibited greater taurine uptake from the basolateral side. Transepithelial taurine flux was in the direction of apical to basolateral in the LLC-PK1 monolayers. There was no net transepithelial movement of taurine in the MDCK monolayers. Efflux of taurine from the apical and the basolateral membrane surfaces of LLC-PK1 cell monolayers was stimulated by external-alanine but not L-alanine. Efflux of taurine from MDCK cell monolayers was stimulated by-alanine on the basolateral surface. While the competitive inhibitor guainidinoeithane sulfonate (GES) competitively inhibited taurine uptake to a similar degree on the apical and basolateral surface of LLC-PK1 cell monolayers, GES had a more potent inhibitory effect on the basolateral taurine uptake in MDCK cells when compared to its inhibition of apical taurine transport. We conclude that there are characteristic differences in transport of taurine by apical and basolateral surfaces of LLC-PK1 and MDCK cells which may be the consequence of asymmetric distribution or unique structural properties of the taurine transporter.Supported by a grant from the National Institutes of Health (DK 37223), the American Heart Association (92-004470).     

Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone in amino acid loaded rats

Summary In adult female anaestetized rats, the influence of triiodothyronine or dexamethasone on renal amino acid handling was investigated in leucine (20mg/100g b.wt.) or glutamine (45mg/100g b.wt.) loaded animals. Bolus injections of both amino acids were followed by temporary increase in fractional excretion of the administered amino acids as well of the endogenous amino acids which were not administered.Under load conditions (leucine and glutamine), dexamethasone treatment (60 g/100 g b.wt. for 3 days, i.p. once daily) was followed by a stimulation of renal amino acid reabsorption. The increase in fractional amino acid excretion after amino acid load was significantly lower than in untreated rats. The effect of triiodothyronine (20,g/1008 b.wt. for 3 days, i.p. once daily) was different in leucine and glutamine loaded animals: after leucine bolus injection a comparable stimulatory effect as shown for dexamethasone could be demonstrated, but after glutamine administration the stimulatory action of T3 was masked. T3 even increases fractional amino acid excretion in glutamine loaded rats as a sign of enhanced house-keeping in the renal tubular cells. These results confirm previous findings and indicate different effects of both hormones on the renal handling of amino acids.