Uracil-induced down-regulation of the yeast uracil permease.
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In Saccharomyces cerevisiae the FUR4-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides. (+info)
Identification of the amine-polyamine-choline transporter superfamily 'consensus amphipathic region' as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to Gabp lacking Cys.
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The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776]. (+info)
A spectroscopic assay for the analysis of carbohydrate transport reactions.
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A carbohydrate-transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or systems with high-affinity constants for sugars. (+info)
Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces.
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Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction. (+info)
Metabolic signals trigger glucose-induced inactivation of maltose permease in Saccharomyces.
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Organisms such as Saccharomyces capable of utilizing several different sugars selectively ferment glucose when less desirable carbon sources are also available. This is achieved by several mechanisms. Glucose down-regulates the transcription of genes involved in utilization of these alternate carbon sources. Additionally, it causes posttranslational modifications of enzymes and transporters, leading to their inactivation and/or degradation. Two glucose sensing and signaling pathways stimulate glucose-induced inactivation of maltose permease. Pathway 1 uses Rgt2p as a sensor of extracellular glucose and causes degradation of maltose permease protein. Pathway 2 is dependent on glucose transport and stimulates degradation of permease protein and very rapid inactivation of maltose transport activity, more rapid than can be explained by loss of protein alone. In this report, we characterize signal generation through pathway 2 using the rapid inactivation of maltose transport activity as an assay of signaling activity. We find that pathway 2 is dependent on HXK2 and to a lesser extent HXK1. The correlation between pathway 2 signaling and glucose repression suggests that these pathways share common upstream components. We demonstrate that glucose transport via galactose permease is able to stimulate pathway 2. Moreover, rapid transport and fermentation of a number of fermentable sugars (including galactose and maltose, not just glucose) are sufficient to generate a pathway 2 signal. These results indicate that pathway 2 responds to a high rate of sugar fermentation and monitors an intracellular metabolic signal. Production of this signal is not specific to glucose, glucose catabolism, glucose transport by the Hxt transporters, or glucose phosphorylation by hexokinase 1 or 2. Similarities between this yeast glucose sensing pathway and glucose sensing mechanisms in mammalian cells are discussed. (+info)
Effect of uncouplers on "downhill" beta-galactoside transport in energy-depleted cells of Escherichia coli.
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Galactoside permease-containing cells of Escherichia coli can be depleted of energy reserves so that the "downhill" cellular hydrolysis of o-nitrophenyl-beta-d-galactopyranoside (ONPG) no longer takes place. Treatment of such energy-depleted cells with proton-conducting agents such as carbonylcyanide m-chlorophenylhydrazone results in stimulation of ONPG transport. The same agents lower transport of non-energy-depleted cells towards the same levels that result from stimulation of the energy depleted cells. Of course, these agents prevent "uphill" accumulation against a concentration gradient under all conditions. Since uncouplers allow normal and energy-depleted cells to assume the same facilitated transport capability, these results lend support to the chemiosmotic hypothesis of Mitchell that comigration of charge is necessary for the transport of neutral galactosides. Our results imply that a potential favorable to transport is maintained by metabolism in non-energy-depleted cells, whereas an unfavorable potential is developed in the initial instant of time when energy-depleted cells are given ONPG. (+info)
Sugar recognition mutants of the melibiose carrier of Escherichia coli: possible structural information concerning the arrangement of membrane-bound helices and sugar/cation recognition site.
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Melibiose carrier mutants, isolated by growing cells on melibiose plus the non-metabolizable competitive inhibitor thiomethyl-beta-galactoside (TMG), were studied to determine sugar and cation recognition abnormalities. Most of the mutants show good transport of melibiose but have lost the recognition of TMG. In addition, most mutants show little or no transport of lactose. Cation recognition is also affected as all of these mutants have lost the ability to transport protons with melibiose. The amino acids causing these mutations were determined by sequencing the melB gene on the plasmid. The mutations were located on helices I, IV, VII, X and XI. We propose that these five helices are in proximity with each other and that they line the sugar/cation transport channel. (+info)
Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity.
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The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated. (+info)