Control of the synthesis, activity, and turnover of enzymes of sulfur metabolism in Neurospora crassa

The synthesis of aryl sulfatase, choline-O-sulfate permease, and two distinct sulfate permeases are repressed by methionine, but the activity of these enzymes is not subject to feedback inhibition. The permease species, but not aryl sulfatase, are also regulated by dynamic turnover, displaying a functional half-life of approximately 2 hr. The rate of turnover of these permeases is not influenced by the presence of the end product, methionine. Development of sulfate permease activity occurs only by de novo synthesis which requires both a lifting of methionine repression and a functional cys-3 product. The turnover system for sulfate permease is not present in dormant conidia but appears to be synthesized relatively rapidly during germination. Preexisting conidial sulfate permease is lost by turnover during germination and outgrowth into the mycelial phase, during which both permease species are synthesized anew, although the high affinity system contributes most of the total activity in growing mycelia.     

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.     

Regulation of sulfate transport in neurospora by transinhibition and by inositol depletion

Neurospora possesses two distinct sulfate transport systems, a low-affinity form (Permease I) which is the only type found in conidia, and a second species (Permease II) which predominates during the mycelial stage. Although methionine represses the synthesis of both of these permeases, inorganic sulfate only partially represses the mycelial form and does not affect the synthesis of Permease I. Both transport systems are also regulated by transinhibition. The transinhibition which occurs in mycelia is not due to an intracellular pool of inorganic sulfate, but is instead exerted by an early intermediate of the sulfate assimilatory pathway.The development of functional sulfate transport activity depends upon genetic and metabolic events which affect the cell membrane. The synthesis of sulfate permease activity in the inos mutant requires an exogenous supply of inositol. The effect of the cot mutant, which is thought to interfere with membrane synthesis, also prevents the development of sulfate permease at the restrictive temperature. The maintenance of pre-existing functional sulfate permease activity apparently also requires a continuous renewal of membrane components since withdrawal of inositol from inos mutants results in a rapid inactivation of transport activity.     

Regulation of sulfate metabolism in Neurospora crassa: Transport and accumulation of glucose 6-sulfate

Neurospora crassa can utilize glucose 6-sulfate as its sole sulfur source, although this compound cannot serve as a carbon source for this organism. Neurospora possesses a transport system capable of glucose 6-sulfate uptake; the system is energy dependent, is inhibited by extracellular sulfate, and is clearly distinct from the permeases responsible for the uptake of glucose and those for sulfate transport. The metabolism of glucose 6-sulfate apparently involves its transport as an intact molecule, followed by a slow intracellular hydrolysis. Methionine, which represses the synthesis of a number of enzymes of sulfur anabolism, also represses the synthesis of the transport system responsible for glucose 6-sulfate uptake. A regulatory gene, cys-3, which controls the synthesis of aryl sulfatase, choline sulfatase, choline-O-sulfate permease, and two distinct permease species, also regulates the permease for glucose 6-sulfate.     

New Method for Study of Peptide Transport in Bacteria

The transport system for glycylmethionine in Escherichia coli B and Salmonella typhimurium LT2 was examined by a new approach which may be applied to other types of exogenous materials. Physiological auxotrophs were prepared by growing wild strains in a methionine-containing medium to repress the methionine biosynthetic enzymes. Immediate protein synthesis was shown to take place in such physiological auxotrophs only in the presence of either exogenous methionine or a methionine peptide, e.g., glycylmethionine. Protein synthesis was dependent on glycylmethionine taken up by the cell and was indicated by assaying for the inducible enzyme lysine decarboxylase at 5- to 15-min intervals. Uptake was studied by using low concentrations of glycylmethionine, therefore making uptake by permease the limiting step in incorporation of methionine into protein, and by addition of competitor peptides to media containing saturating concentrations of glycylmethionine. Lysine decarboxylase activity in S. typhimurium LT2 was about 80 times that present in E. coli B. Glycylmethionine transport had a K(m) of the order of 1 muM in S. typhimurium. Structural specificities observed for peptide transport by other workers were confirmed for E. coli B. Competitive inhibition of glycylmethionine uptake by dipeptides was observed in E. coli.     

Cooperative binding of the sugar substrates and allosteric regulatory protein (enzyme IIIGlc of the phosphotransferase system) to the lactose and melibiose permeases in Escherichia coli and Salmonella typhimurium

An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-beta-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-alpha-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-beta-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-alpha-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme III(Glc) to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme III(Glc)) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme III(Glc) to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme III(Glc), the postulated regulatory protein of the PTS.     

Deficiency in methionine adenosyltransferase resulting in limited repressibility of methionine biosynthetic enzymes in Aspergillus nidulans

Summary In Aspergillus nidulans methionine can be metabolized to cysteine. Mutants blocked in this pathway were selected and divided into three groups representing three separate loci: mecA, mecB and mecC. mecC13 mutant possesses a low level of methionine adenosyltransferase and shows a limited extent of methionine-caused repression of three enzymes of the methionine biosynthetic pathway: sulfate permease, sulfite reductase and 0-acetylhomoserine sulfhydrylase. Intracellular pools of methionine do not differ markedly in the mutant and in wild type, while the S-adenosylmethionine (SAM) pool is decreased in the mutant. Methionine adenosyltransferase was found to be inducible by methionine, SAM is postulated to be involved in regulation of methionine biosynthetic enzymes in A. nidulans. Differences in regulation of methionine biosynthesis in A. nidulans, Escherichia coli and Saccharomyces cerevisiae are discussed.     

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.     

Kinetics of active alpha-glucoside transport in Saccharomyces cerevisiae

alpha-Glucosides are the most abundant fermentable sugars in the industrial applications of Saccharomyces cerevisiae, and the active transport across the plasma membrane is the rate-limiting step for their metabolism. In this report we performed a detailed kinetic analysis of the active alpha-glucoside transport system(s) present in a wild-type strain, and in strains with defined alpha-glucoside permeases. Our results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and alpha-methylglucoside. The maltose permease encoded by the MAL21 gene showed a high affinity (K(m) approximately 5 mM) for maltose, and a low affinity (K(m) approximately 90 mM) for trehalose. On the other hand, the alpha-glucoside permease encoded by the AGT1 gene had a high affinity (K(m) approximately 7 mM) for trehalose, a low affinity (K(m) approximately 18 mM) for maltose and maltotriose, and a very low affinity (K(m) approximately 35 mM) for alpha-methylglucoside.     

Ureidosuccinic Acid Uptake in Yeast and Some Aspects of Its Regulation

Ureidosuccinic acid (USA) is an intermediary product in pyrimidine biosynthesis. When proline was the sole nitrogen source, USA uptake occurred; however, when ammonium sulfate or glutamic acid was the nitrogen source, uptake was inhibited. Thus, a ura2 strain which does not synthesize USA would not grow when this substance was supplied on an ammonium sulfate or glutamic acid medium. Mutants are described in which uptake was constitutive on such a medium. Permeaseless mutants for USA have been found, and evidence is presented for permease specificity. It is shown that all constitutive mutants use the same transport system that is missing in the permeaseless mutant. These mutants are constitutive for two permeases: the specific USA permease and the general amino acid permease. The transport system studied here, like the general amino acid transport system, is regulated by nitrogen metabolism. These facts and others suggest that our permease constitutive mutants are impaired in nitrogen metabolism.     

Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.

Escherichia coli forms three permeases that can transport the amino acid tryptophan: Mtr, AroP, and TnaB. The structural genes for these permeases reside in separate operons that are subject to different mechanisms of regulation. We have exploited the fact that the tryptophanase (tna) operon is induced by tryptophan to infer how tryptophan transport is influenced by the growth medium and by mutations that inactivate each of the permease proteins. In an acid-hydrolyzed casein medium, high levels of tryptophan are ordinarily required to obtain maximum tna operon induction. High levels are necessary because much of the added tryptophan is degraded by tryptophanase. An alternate inducer that is poorly cleaved by tryptophanase, 1-methyltryptophan, induces efficiently at low concentrations in both tna+ strains and tna mutants. In an acid-hydrolyzed casein medium, the TnaB permease is most critical for tryptophan uptake; i.e., only mutations in tnaB reduce tryptophanase induction. However, when 1-methyltryptophan replaces tryptophan as the inducer in this medium, mutations in both mtr and tnaB are required to prevent maximum induction. In this medium, AroP does not contribute to tryptophan uptake. However, in a medium lacking phenylalanine and tyrosine the AroP permease is active in tryptophan transport; under these conditions it is necessary to inactivate the three permeases to eliminate tna operon induction. The Mtr permease is principally responsible for transporting indole, the degradation product of tryptophan produced by tryptophanase action. The TnaB permease is essential for growth on tryptophan as the sole carbon source. When cells with high levels of tryptophanase are transferred to tryptophan-free growth medium, the expression of the tryptophan (trp) operon is elevated. This observation suggests that the tryptophanase present in these cells degrades some of the synthesized tryptophan, thereby creating a mild tryptophan deficiency. Our studies assign roles to the three permeases in tryptophan transport under different physiological conditions.     

Interrelated regulation of sulphur-containing amino-acid biosynthetic enzymes and folate-metabolizing enzymes in Aspergillus nidulans

In Aspergillus nidulans homocysteine can be metabolized both to cysteine and methionine. Mutants impaired in the main pathway of cysteine synthesis or in the sulphate assimilation pathway show a low pool of glutathione and elevated levels of homocysteine synthase and of the homocysteine-to-cysteine pathway enzymes. On the other hand, the level of methionine synthase and other enzymes of folate metabolism is depressed in these mutants. This anticoordinated regulation provides a mechanism controlling the partition of homocysteine between the two diverging pathways. Homocysteine synthase was found derepressed, along with folate enzymes, in a strain carrying a mutation which suppresses mutations in metA, metB and metG genes. These results indicate that homocysteine synthase can be regarded as the enzyme of an alternative pathway of methionine synthesis and strongly suggest that the regulatory mechanisms governing sulphur-containing amino acid and folate metabolisms are interrelated.     

Identification and functional characterization of a Xenorhabdus nematophila oligopeptide permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA(1), oppA(2), or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Phospho-β-glucosidases and β-Glucoside Permeases in Streptococcus, Bacillus, and Staphylococcus

The presence of phospho-beta-glucosidases and beta-glucoside permeases was found in strains of Streptococcus, Bacillus, and Staphylococcus. In streptococci, the phospho-beta-glucosidase activity depends on the antigenic group. The highest activity was found in strains of group D. In group D strains, phospho-beta-glucosidase activity is induced by beta-methyl glucoside and cellobiose but not by thiophenyl beta-glucoside (TPG). With the exception of four strains isolated in Japan, all strains of B. subtilis tested possess an inducible phospho-beta-glucosidase activity, beta-methyl glucoside, cellobiose, and TPG acting as inducers. S. aureus strains possess phospho-beta-glucosidase A but not phospho-beta-glucosidase B, whereas most S. albus strains show no detectable phospho-beta-glucosidase activity. The prompt fermentation of beta-methyl glucoside by S. aureus strains could serve as an additional criterion for their differentiation from S. albus. A comparative investigation of the active uptake of (14)C-TPG showed that a Streptococcus group D strain and a B. subtilis strain posses two inducible permeases with characteristics similar to the beta-glucoside permeases I and II of Enterobacteriaceae. In S. aureus, TPG is accumulated by a constitutive permease with high affinity for aromatic beta-glucosides and glucose. The active uptake of TPG by S. aureus appears to depend on the activity of the phosphoenol pyruvate-dependent phosphotransferase system.     

Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast.

In Saccharomyces cerevisiae, amino acid permeases are divided into two classes. One class, represented by the general amino acid permease GAP1, contains permeases regulated in response to the nitrogen source. The other class, including the high affinity tryptophan permease, TAT2, consists of the so-called constitutive permeases. We show that TAT2 is regulated at the level of protein stability. In exponentially growing cells, TAT2 is in the plasma membrane and also accumulates in internal compartments of the secretory pathway. Upon nutrient deprivation or rapamycin treatment, TAT2 is transported to and degraded in the vacuole. The ubiquitination machinery and lysine residues within the NH(2)-terminal 31 amino acids of TAT2 mediate ubiquitination and degradation of the permease. Starvation-induced degradation of internal TAT2 is blocked in sec18, sec23, pep12, and vps27 mutants, but not in sec4, end4, and apg1 mutants, suggesting that, upon nutrient limitation, internal TAT2 is diverted from the late secretory pathway to the vacuolar pathway. Furthermore, our results suggest that TAT2 stability and sorting are controlled by the TOR signaling pathway, and regulated inversely to that of GAP1.