Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.     

Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae.

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.     

Genetic relationships among actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin

Summary A polyphasic taxonomic study was undertaken to establish the genetic and phenotypic relationships among six actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin. Chemotaxonomic studies reveal that all have Type I cell walls. Gas chromatography (GC) of fatty acid methyl esters revealed patterns consistent for strains ofStreptomyces with 160 and 150anteiso predominating. Principal component analysis of GC data revealed distinct profiles for each culture. Reciprocal DNA homology studies atT _m -25 showed the rapamycin-producing strain and one FK506-producing strain to have 38–50% homology with the type strain ofStreptomyces hygroscopicus (ATCC 27438). The remaining strains exhibited 6–17% homology. To further explore the relationships among these strains all were probed for the presence of anO-methyltransferase gene specific to this biosynthetic pathway. Among the strains of interest, onlyStreptomyces hygroscopicus subsp.yakushimaensis, the patent strain for FK520/FK523, failed to hybridize with the probes.     

Regulation of dipeptide transport in Saccharomyces cerevisiae by micromolar amino acid concentrations.

Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine- and m-fluorophenylalanine-containing toxic dipeptides. We examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides. A number of these amino acids, at concentrations as low as 1 microM (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine. Sensitivity to ethionine and m-fluorophenylalanine remained high under either set of conditions. The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L-[3H]leucine accumulation. This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan. Comparable increases in peptidase activity were not apparent in crude cell extracts from tryptophan-induced cultures. We concluded that S. cerevisiae possesses a specific mechanism for the induction of dipeptide transport that can respond to very low concentrations of amino acids.     

Mutations in Saccharomyces cerevisiae which confer resistance to several amino acid analogs.

Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline. All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake. In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1. Both raa4 and gap1 prevent uptake of [3H]leucine in liquid cultures. The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Pathological change of articular cartilage in the mandibular head treated with immunosuppressant FK 506

While several reports have documented immunosuppressant-induced osteoporosis, the exact mechanism of the pathological change of the joint remains to be clarified. In the present study, we have demonstrated the pathological change of the articular cartilage in the mandibular head of five Sprague-Dawley rats administered with the immunosuppressant FK 506 for 28 days. Three-dimensional micro-computed tomography of the mandibular heads in treated rats showed a significant decrease in trabecular bone volume compared to control rats. Histological observation revealed atrophic change of the articular cartilage. Immunohistological observation using anti-proliferative cell nuclear antibody (PCNA), type I, II, and type X collagen antibodies showed significantly decreased proliferation and differentiation of chondrocytes in the articular cartilage compared with the control group (p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining revealed no significant difference in the numbers of osteoclasts at the chondro-osseous junction. Thus, FK 506 administration inhibited chondrogenic cell proliferation and differentiation and might cause osteoporotic change of subcartilage trabecular bone that subsequently forms in the mandibular head.     

Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506)

The current off-patent state of tacrolimus (FK506) has opened the hunting season for new generic pharmaceutical formulations of this immunosuppressant. This fact has boosted the scientific and industrial research on tacrolimus for the last 5 years in order to improve its production. The fast discovery of tacrolimus producer strains has generated a huge number of producers, which presents the biosynthetic cluster of FK506 as a high promiscuous genetic region. For the first time, the current state-of-the-art on the tacrolimus biosynthesis, production improvements and drug purification is reviewed. On one hand, all the genes involved in the tacrolimus biosynthesis, in addition to the traditional PKS/NRPS, as well as their regulation are analysed. On the other hand, tacrolimus direct and indirect precursors are reviewed as a straight manner to improve the final yield, which is a current trend in the field. Twenty years of industrial and scientific improvements on tacrolimus production are summarised, whereas future trends are also drafted.     

Purification and amino acid sequence of mating factor from Saccharomyces cerevisiae.

Mating factor is a peptide excreted into the culture fluid by alpha-mating type cells of Saccharomyces cerevisiae X-2180 1B. The purification of the mating factor was carried out by ion exchange chromatography on phosphocellulose and Amberlite IRC 50 columns, followed by gel filtration on a Sephadex LH 20 column. The factor thus prepared was a peptide composed of Lys1, His1, Trp2, Gln2, Pro2, Gly1, Met1, Leu2 and Tyr1, and was able to induce morphological changes on alpha-mating type cells at a concentration of 5 pg/ml. The amino acid sequence of the mating factor was determined by the manual Edman degradation method using intact mating factor and its thermolytic peptides. The C-terminal amino acid residue was determined by digesting the factor with carboxypeptidase A. The complete amino acid sequence of the mating factor was established to be as follows: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr.     

The complete amino acid sequence of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae

The crystallizable cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63 000). Its primary structure was established using peptide sequences from four different digests of the native and citraconylated enzyme with trypsin, cyanogen bromide and staphylococcal protease. The oligonucleotide sequence of the structural gene was used as a template for the final alignment of the various peptides in the correct order.     

Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.     

Free intracellular amino acid pools during autonomous oscillations in Saccharomyces cerevisiae

In the present work dynamic changes of free intracellular amino acid pools during autonomous oscillations of Saccharomyces cerevisiae were quantified in glucose-limited continuous cultivations. At a dilution rate of D = 0.22 h(-1) cyclic changes with a period of 120 min were found for many variables such as carbon dioxide production rate, dissolved oxygen, pH, biomass content, and various metabolite concentrations. On the basis of the observed dynamic patterns, free intracellular amino acids were classified to show oscillatory, stationary, or chaotic behavior. Amino acid pools such as serine, alanine, valine, leucine, or lysine were subjected to clear oscillations with a frequency of 120 min, identical to that of other described cultivation variables, indicating that there is a direct correlation between the periodic changes of amino acid concentrations and the metabolic oscillations on the cellular level. The oscillations of these amino acids were unequally phase-delayed and had different amplitudes of oscillation. Accordingly, they exhibited different patterns in phase plane plots vs. intracellular trehalose. Despite the complex and marked metabolic changes during oscillation, selected intracellular amino acids such as histidine, threonine, isoleucine, or arginine remained about constant. Concentrations of glutamate and glutamine showed a chaotic behavior. However, the ratio of glutamate to glutamine concentration was found to be oscillatory, with a period of 60 min and a corresponding figure eight-shaped pattern in a plot vs. trehalose concentration. Considering the described diversity, it can be concluded that the observed periodic changes are neither just the consequence of low or high rates of protein biosynthesis/degradation nor correlated to changing cell volumes during oscillation. The ratio between doubling time (189 min) and period of oscillation of intracellular amino acids (120 min) was 1:6. The fact that there is a close relationship between doubling time and period of oscillation underlines that the described autonomous oscillations are cell-cycle-associated.     

Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.