Mutations in Neurospora crassa which affect multiple amino acid transport systems.

Characterization of a double mutant, his-6: hgu-4, which is unable to utilize L-histidyl-glycine as a source of histidine has revealed a new locus on linkage group V. The hgu-4 genotype results in a generalized reduced transport activity for amino acids, with a concomitant increased resistance to amino acid analogs. Transport rates and analog resistance for amino acids by this mutant are compared to the previously reported transport deficient mutants fpr-1, nap and un-3. Transport of L-aspartate as a function of temperature is examined in a variety of transport deficient strains in an attempt to explain the mode of action of mutation which pleiotropically affect several genetically and biochemically distinct amino acid transport systems.     

Vba4p,a vacuolar membrane protein,is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Amino acid transport during development of Neurospora crassa conidia: Substrate repression

The increasing amino acid transport activity which occurs during germination of Neurospora crassa is repressed by substrate amino acid. This repression acts on the transport systems similarly to competition in that amino acids within a specific transport class (e.g., basic) repress that system. Repression of the other system (neutral-aromatic) by that amino acid is shown to be repression of the general transport system. The level of repression and the rate of derepression after removal of the amino acid appear to depend on the nonrepressed level and rate. The extent of repression caused by increasing the concentration of the amino acid is shown to be different for two amino acids. A mutant deficient in developmental transport for arginine and phenylalanine contains two mutations. The mutation affecting phenylalanine transport maps on linkage group III and results in an accumulation of phenylalanine in the medium, thus repressing the development of this transport activity.This work was supported in part by a National Institutes of Health, U.S. Public Health Service Traineeship in Genetics (2-T01-GM1316).     

AMINO ACID TRANSPORT IN NITZSCHIA OVALIS ARNOTT1,2

The marine diatom Nitzschia ovalis possesses at, least 3 amino acid uptake systems, specific for transport of acidic, polybasic, and, neutral amino acids. Maximum uptake velocity (V_max) for each, site is inversely related to the nitrogen content of the cell, and to the nitrogen available in the culture medium. Transport, of polybasic amino acids occurs throughout the course of growth in batch, culture, but the V_max increases dramatically as the culture ages and nitrogen/cell reaches a low value. K_s does not, change significantly. Acidic and neutral amino acids are taken up only by cells harvested from nitrogen-poor culture. It appears that amino acid transport is repressed by high concentrations of nitrogen in the medium. Under natural conditions, where nitrogen concentrations are low, the contribution of amino acid uptake to the nitrogen economy of Nitzschia populations may be significant.     

Redox regulation of free amino acid levels in Arabidopsis thaliana

Abiotic stresses induce oxidative stress, which modifies the level of several metabolites including amino acids. The redox control of free amino acid profile was monitored in wild‐type and ascorbate or glutathione deficient mutant Arabidopsis thaliana plants before and after hydroponic treatment with various redox agents. Both mutations and treatments modified the size and redox state of the ascorbate (AsA) and/or glutathione (GSH) pools. The total free amino acid content was increased by AsA, GSH and H₂O₂ in all three genotypes and a very large (threefold) increase was observed in the GSH‐deficient pad2‐1 mutant after GSH treatment compared with the untreated wild‐type plants. Addition of GSH reduced the ratio of amino acids belonging to the glutamate family on a large scale and increased the relative amount of non‐proteinogenic amino acids. The latter change was because of the large increase in the content of alpha‐aminoadipate, an inhibitor of glutamatic acid (Glu) transport. Most of the treatments increased the proline (Pro) content, which effect was due to the activation of genes involved in Pro synthesis. Although all studied redox compounds influenced the amount of free amino acids and a mostly positive, very close (r > 0.9) correlation exists between these parameters, a special regulatory role of GSH could be presumed due to its more powerful effect. This may originate from the thiol/disulphide conversion or (de)glutathionylation of enzymes participating in the amino acid metabolism.     

Derepressed leucine transport activity in Escherichia coli

Transport activity and synthesis of binding protein for the amino acids leucine, isoleucine and valine in E. coli are coordinately controlled by the level of leucine in the growth medium. Spontaneous mutants (dlu) which can utilize D-leucine as a source of L-leucine show derepressed transport activity for the three-branched chain amino acids. The increased transport activity is a result of an increase in the binding protein for these amino acids. Azaleucine-resistant mutants have been isolated which have a defect in leucine transport but normal levels of the binding protein for leucine.     

Peculiarities of amino acid transport inSchizosaccharomyces pombe: Effects of growth medium

Transport systems for amino acids in the wild-type strain ofSchizosaccharomyces pombe are not constitutive. During growth on different media no transport of acidic, neutral and basic amino acids is detectable. To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose. The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth. After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used.l-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.     

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databanks and have been assigned the accession number X78413     

Active transport of l-aspartic acid in Neurospora crassa

Transport of l-aspartic acid in Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The transport activity is dependent on pH and results in accumulation of l-aspartic acid against a gradient. Mutants deficient in transport of l-aspartic acid are described.     

A general amino-acid permease is inducible in Chlorella vulgaris

Glucose or non-metabolizable glucose analogues induce two systems of amino-acid transport in Chlorella vulgaris: an arginine-lysine system and a proline system. An additional third system of amino-acid transport is induced when glucose and an inorganic nitrogen source are present during glucose induction. The transport rates in glucose-NH ₄ ⁺ -treated cells are 10 to 80 times higher than in untreated cells. The transport system shows a rather broad specificity and catalyses the transport of at least ten neutral and acidic amino acids. Three of these amino acids (l-alanine, l-serine and glycine) are transported by the proline system as well. The system is specific for l-amino acids and has a pH optimum between 5 and 6. Transport by this system seems to be active, since amino acids are accumulated inside the cells.     

Incorporation of 14C-amino acids by malarial plasmodia (Plasmodium iophurae). VI. Changes in the kinetic constants of amino acid transport during infection

The majority (10 of 17) of amino acids tested entered the mature duck erythrocyte by a saturable, non-uphill transport system, whereas for the erythrocyte-free malarial parasite, Plasmodium lophurae, the converse was true: most amino acids entered the parasite by simple diffusion. Only five amino acids (glutamic and aspartic acids, cysteine, lysine, arginine) showed mediated entry into P. lophurae. The pattern of mediated amino acid transport into the duck erythrocyte was altered upon infection, e.g., either entry was by diffusion or there was a reduced affinity for the amino acid. Transport characteristics similar to those found in the malaria-infected erythrocyte were produced by treating normal duck red cells with a cell-free extract of malaria-infected erythrocytes and quinine (a depressor of red cell ATP). It is suggested that depletion of host cell ATP as well as elaboration of as yet unidentified substances by the parasite promote the changes in permeability seen in the malaria-infected cell.     

The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.     

gamma-Glutamyl amino acids. Transport and conversion to 5-oxoproline in the kidney

Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of 35S after incubation of the slices in media containing gamma-glutamyl methionine [35S]sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices.     

Hormonal regulation of the system a amino acid transport adaptive response mechanism in a kidney epithelial cell line (MDCK)

When mamalian cells are starved for amino acids, the activity of the A amino acid transport system increases, a phenomenon called adaptive regulation. We have examined the effects of those factors which support Madin-Darby canine kidney (MDCK) cell growth in a defined medium on the derepression of System A activity. Of the five factors which supported MDCK cell growth, insulin was found to be an absolute requirement for derepression. In contrast, PGE₁ was a negative controlling factor for the transport system. Growth of MDCK cells in the absence of PGE₁ resulted in elevated System A activity which derepressed poorly upon amino acid starvation. Kinetic analysis of α-(methylamino) isobutyric acid (mAIB) uptake as a function of substrate concentration showed that the elevated A activity observed when cells were grown in the absence of PGE₁ was kinetically similar to the activity induced by starvation for amino acids. Transport of mAIB by amino-acid-fed cells grown in the presence of PGE₁ was characterized by a linear Eadie-Hofstee graph and by a relatively low Vmax. Transport by cells starved for amino acids or by cells grown in the absence of PGE₁ was characterized by biphasic kinetics for mAIB transport and by elevated Vmax values. An influence of growth factors on the inactivation of derepressed A activity was also observed. In the presence of cycloheximide the rate of loss of A activity in amino-acid-starved cells was 1/4–1/2 that of amino-acid-fed cells. Insulin slowed inactivation in the absence of most amino acids in a protein-synthesis-independent manner, but insulin did not influence the more rapid inactivation observed in amino-acid-fed cells. These results indicate that the level of System A activity observed in response to regulation by amino acids represents a balance between carrier synthesis and inactivation, which can be positively or negatively influenced by growth factors.     

A candidate mouse model for Hartnup Disorder deficient in neutral amino acid transport

The mutant mouse strain HPH2 (hyperphenylalaninemia) was isolated after N-ethyl-N-nitrosourea (ENU) mutagenesis on the basis of delayed plasma clearance of an injected load of phenylalanine. Animals homozygous for the recessive hph2 mutation excrete elevated concentrations of many of the neutral amino acids in the urine, while plasma concentrations of these amino acids are normal. In contrast, mutant homozygotes excrete normal levels of glucose and phosphorus. These data suggest an amino acid transport defect in the mutant, confirmed in a small reduction in normalized values of ¹⁴C-labeled glutamine uptake by kidney cortex brush border membrane vesicles (BBMV). The hyperaminoaciduria pattern is very similar to that of Hartnup Disorder, a human amino acid transport defect. A subset of Hartnup Disorder cases also show niacin deficiency symptoms, which are thought to be multifactorially determined. Similarly, the HPH2 mouse exhibits a niacin-reversible syndrome that is modified by diet and by genetic background. Thus, HPH2 provides a candidate mouse model for the study of Hartnup Disorder, an amino acid transport deficiency and a multifactorial disease in the human. Received: 16 May 1996 / Accepted: 25 September 1996     

The ArsQ permease and transport of the antibiotic arsinothricin

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.