Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids

Summary Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.     

A lethal allele at the putative regulatory locus, cpc-1, of cross-pathway control in Neurospora crassa

Summary A lethal allele at the putative regulatory locus, cpc-1, of cross-pathway control in Neurospora crassa was discovered by genetic analysis. cpc-1 ^j-5 is viable only in the presence of a second mutation, slo, causing slow growth. The detection of a lethal allele at a regulatory locus is a rare event and points to the physiological importance of the regulatory circuit concerned, namely the cross-pathway or general control of amino acid biosynthetic enzymes in lower eukaryotes.     

cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle.

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.     

The Tsc/Rheb signaling pathway controls basic amino acid uptake via the Cat1 permease in fission yeast

The Tsc/Rheb signaling pathway plays critical roles in the control of growth and cell cycle. Studies in fission yeast have also implicated its importance in the regulation of amino acid uptake. Disruption of tsc2 ⁺, one of the tsc ⁺ genes, has been shown to result in decreased arginine uptake and resistance to canavanine. A similar effect is also seen with other basic amino acids. We have identified a permease responsible for the uptake of basic amino acids by genetic complementation and disruption. SPAC869.11 (termed Cat1 for cationic amino acid transporter) contains 12 predicted transmembrane domains and its overexpression in wild type fission yeast leads to the increased uptake of basic amino acids and sensitivity to canavanine. Disruption of cat1 ⁺ in the Δtsc2 background interfered with the suppression of the canavanine-resistant phenotype of Δtsc2 mutants by a dominant negative Rheb. In Δtsc2 mutant strains, the amount of Cat1 was not altered, but instead was mislocalized. This mislocalization was suppressed by the expression of dominant negative Rheb. In addition, we found that the loss of the E3 ubiquitin ligase, Pub1, also restores proper localization. These results provide a crucial link between Tsc/Rheb signaling and the regulation of the basic amino acid permease in fission yeast.     

Effect of N-methyl-N′-Nitro-N-nitrosoguanidine on amino acid incorporation into mucosal protein of rat stomach

Administration of the carcinogenic N-nitroso compound, N-methyl-N′-nitro-N-nitrosoguanidine in drinking water (0.5 mg/mL) to male Wistar rats for 1 week caused impairment of in vivo and in vitro incorporation of [¹⁴C]leucine into stomach mucosal protein. This impairment gradually returned to normal after 4 weeks. Uptake of [¹⁴C]leucine into mucosal protein was significantly inhibited after in vitro treatment of stomach mucosa with the carcinogen. Addition of the N-nitroso compound in a cell-free system using postmitochondrial supernatant prepared from stomach mucosa also showed inhibition of amino acid incorporation. Using a more defined system consisting of purified polyribosome from stomach mucosa and pH 5 enzyme fraction derived from liver it was further demonstrated that the carcinogen purturbed protein synthesizing ability of polyribosome, under both in vivo and in vitro treatment conditions. In these respects this carcinogen has similar action on the target tissue of stomach as in the liver, although the in vivo effect may be related more to toxicity than carcinogenicity.     

The relA locus and the regulation of lysine biosynthesis in Escherichia coli

Summary The allelic state of relA influences the phenotype of Escherichia coli strains carrying the lysA22 mutation: lysA22 relA strains are Lys^– where lysA22 relA ⁺ strains grow (slowly) in the absence of lysine. This physiological effect has been related to an effect of the expression of the relA locus on the regulation of lysine biosynthesis. The fully derepressed levels of some lysine enzymes (aspartokinase III, aspartic semialdehyde dehydrogenase, dihydrodipicolinate reductase) are observed under lysine limitation only in rel ⁺ strains. And the induction of DAP-decarboxylase by DAP is much higher in rel ⁺ than in rel ^– strains when an amino acid limitation of growth is also realised. These results are in agreement with the hypothesis of Stephens et al. (1975) on a possible role of the stringent regulation as a general signal for amino acid deficiency.     

Glucose and amino acid metabolism in chick telencephalon slices: Changes with incubation conditions and animals' development

Glucose and amino acid metabolism in 1- and 30-day-old chick telencephalon slices was studied in two incubation media in the presence or in the absence of a continuous oxygenation. Medium 1 has a composition and a tonicity similar to cerebrospinal fluid, medium 2 is hypertonic and does not contain any K⁺ ions. The incorporation of glucose carbon into amino acids and the distribution of radioactivity between the different amino acids are close to the ones observed in the chick brain in vivo only when the slices are incubated in medium 1, with oxygen at 30 days and without oxygen for the 1-day-old chick. It also appears that if oxygenation is necessary for incubation of mature brain tissue in vitro, the absence of the medium oxygenation is more suitable for the study of glucose metabolism in 1-day-old chick brain slices.     

Differential effect of amino acid starvation on polysome decay in escherichia coli

In arelA⁺ strain ofE. coli starved separately for each of four required amino acids, the intracellular concentration of polysomes decreases as a function of time in all cases: very rapidly in the absence of arginine or leucine, slowly in the absence of threonine or histidine. In a starved isogenicrelA strain, the polysome level is either totally stable or else drops slowly. The decrease in the level, when it occurs, does not significantly affect the polysome size distribution. Models for polysome metabolism in amino acid starved cells are discussed.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.     

The stimulation of amino acid incorporation into the protein of unfertilized sea urchin eggs by gamma-irradiation

Control and γ-irradiated sea urchin (Arbacia punctulata) eggs were incubated with H³-leucine. No incorporation of the amino acid into the proteins of most unirradiated eggs was detectable by autoradiography. All irradiated eggs incorporated the amino acid and the amount of autoradiographic labelling varied greatly from cell to cell.     

Phenotypic expression of epigenetic determinant [ISP +] in Saccharomyces cerevisiae depends on the combination of sup35 and sup45 mutations

Translation fidelity in Saccharomyces yeasts is determined by genetic and epigenetic (prion) factors. A study was made of S. cerevisiae strains containing the nonchromosomal determinant [ISP ⁺], described earlier. Some of its properties suggest that [ISP ⁺] is a prion. [ISP ⁺] is expressed phenotypically as an antisuppressor of two sup35 mutations and can be cured with guanidine chloride (GuHCl). It was shown that sup35 mutants containing [ISP ⁺] carried additional sup45 mutations. These mutations caused amino acid substitutions in different regions of translation termination factor eRF1, encoded by SUP45. Strains bearing the sup35-25 mutation contained the sup45 mutation that caused amino acid substitution at position 400 of eRF1; strains bearing sup35-10 contained the mutation that altered eRF1 at position 75. Thus, the antisuppressor phenotype of the [ISP ⁺] strains proved to depend on the interaction of sup35 and sup45 mutations, as well as on the GuHCl-curable epigenetic determinant. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 844–849. The article was translated by the authors.     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide