Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation.

Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.

Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 µg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 µg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and 50 nm) was investigated at a very low dose for 60 days (40 ng gold/fish/day). Mitochondrial dysfunctions appeared in brain and muscle for both tested sizes. In conclusion, at low environmental doses, dietary or waterborne heavy metals impinged on zebrafish tissue mitochondrial respiration. Due to its incredible simplicity avoiding tedious and time-consuming mitochondria isolation, our one-pot method allowing brain respiratory analysis should give colleagues the incentive to use zebrafish brain as a model in bioenergetics. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio).

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13 microg of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

Environmental study of subunit i, a F(o) component of the yeast ATP synthase.

The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants. The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents. Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex. Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase.

Evidence for a dynamic role for proline376 in the purine-cytosine permease of Saccharomyces cerevisiae.

The purine-cytosine permease (PCP), a carrier located in the plasma membrane of Saccharomyces cerevisiae, mediates the active transport of purine (adenine, guanine and hypoxanthine) and cytosine into the cell. Previous studies [Ferreira, T, Brèthes, D., Pinson, B., Napias, C. & Chevallier, J. et al. (1997) J. Biol. Chem. 272, 9697-9702] suggest that the hydrophilic segment 371-377 (-I-A-N-N-I-P-N-) of the polypeptide chain may play a key role in the correct three-dimensional structure of the active carrier. This paper describes the effects of mutations in this particular segment: a four-residue deletion, Delta374-377, and two substitutions, P376G and P376R. The Delta374-377 PCP was expressed in tiny amounts and was totally inactive. When compared with the wild-type, the P376G PCP showed slightly decreased amounts and was able to transport the bases with significantly increased affinity and decreased turnover. The P376R PCP was normally expressed and targeted to the plasma membrane; however, despite a normal number of base-binding sites [1000-1200 pmol.(mg protein)-1], this mutated carrier was completely unable to transport any of its ligands. In addition, the Kd(app) for hypoxanthine binding was completely independent of the pH (within the range 3.5-6.0), showing that the conformational change induced by ligand binding was no longer present. Our results show that the 374-377 segment is essential for the expression and activity of this carrier. They also show that the P376 residue is part of an unusual secondary structure, probably a beta-turn motif, which must play a crucial dynamic role in the translocation process.

Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae. Possible role of Ser272 in the base translocation process.

The purine-cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second-site mutation corresponds to the replacement of the Ser272 residue by Leu. Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease. To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272I, was constructed. Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base-binding step that could account for its global suppressive effect. Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton.

In vitro and in situ intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells lining a polyester fabric.

In order to improve long-term patency of vascular grafts, the promising concept of endothelial cell seeding is actually under investigation. Our laboratory tested a polyester coated with albumin and chitosan which permits a rapid colonization by human umbilical vein endothelial cells (HUVEC) and it seems relevant to test in vitro the expression of adhesive molecules expressed by cells with regard to the inflammatory process. We studied intercellular adhesion molecule-1 (ICAM-1) expression and focused our work on the determination of ICAM-1 sites expressed per adherent cell lining the biomaterial, thus in situ, in comparison to control HUVEC on plastic wells: the results obtained by binding experiments were correlated to flow cytometry analyses and showed that the polyester does not induce a proinflammatory state and that HUVEC covering the structure are able to respond to a stimulus.

Post-translational fate of CAN1 permease of Saccharomyces cerevisiae.

To study the post-translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag. In exponential-phase cells the amount of Can1p is constant, although turnover can be demonstrated. A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein. The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine-grown cells, in which the amount of the protein is markedly lower. This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources. it was demonstrated that Can1p is phosphorylated. Since Can1p expression under the PMA1 promoter is glucose-dependent, the amount of the permease expressed in high-glucose-grown cells is higher than in low-glucose-grown ones. Only a part of the Can1p overexpressed in high-glucose-grown cells is phosphorylated, while in low-glucose-grown cells the phosphorylated form probably represents the majority of Can1p. The permease phosphorylation or dephosphorylation is not related to transinhibition.

Characterization of the Saccharomyces cerevisiae cytosine transporter using energizable plasma membrane vesicles.

The purine-cytosine permease is a carrier localized in the plasma membrane of the yeast Saccharomyces cerevisiae. The energetics of cytosine transport catalyzed by this permease has been studied in an artificial system obtained by fusion between proteoliposomes containing beef heart cytochrome c oxidase and plasma membrane-enriched fractions of a S. cerevisiae strain overexpressing the permease. Upon addition of an energy donor, a proton-motive force (inside alkaline and negative) is created in this system and promotes cytosine accumulation. By using different phospholipids, it is shown that cytosine uptake is dependent on the phospholipids surrounding the carrier. It was demonstrated that the purine-cytosine permease is able to catalyze a secondary active transport of cytosine. By using nigericin and valinomycin, the DeltapH component of the proton-motive force is shown to be the only force driving nucleobase accumulation. Moreover, transport measurements done at two pH values have shown that alkalinization of intravesicular pH leads to a significant increase in cytosine uptake rate. Finally, no specific role of K+ ions on cytosine transport could be demonstrated in this system.

Functional analysis of mutated purine-cytosine permease from Saccharomyces cerevisiae. A possible role of the hydrophilic segment 371-377 in the active carrier conformation.

The purine-cytosine permease (PCP) is an active transporter located in the plasma membrane of the yeast Saccharomyces cerevisiae. This protein mediates purine (adenine, guanine, and hypoxanthine) and cytosine accumulation in the cell by using an electrochemical potential difference in proton as the energy source. Various mutant strains, with altered Kt(app) (apparent Michaelis constant of transport) of uptake for one or several bases, have already been selected. Their cloning and sequencing revealed that three of them presented substitutions in the same region of the putative sequence of the PCP: this region might correspond to the hydrophilic segment 371-377 (I-A-N-N-I-P-N). Two mutants displayed single mutations, resulting in only one amino acid residue change (N377I and N374I, respectively), and the other displayed three amino acid substitutions (I371V, I375V, and N377G). Therefore, to analyze the contribution of individual amino acid changes to the phenotype of the complex mutant, single (N377G) and double (I371V,I375V) mutants were constructed by site-directed mutagenesis. The influence of single mutations in this region was studied by measuring, for adenine, hypoxanthine, and cytosine, the uptake constants on cells and equilibrium binding parameters on plasma membrane-enriched fractions. Uptake and binding constant determinations showed that all the variations observed for the Kt(app) of uptake were correlated with variations of the binding Kd(app) for the corresponding solutes. Thus, our results emphasize the role of the two asparagine residues, located at positions 374 and 377, respectively, in the binding of the bases. In addition, the sole substitution of the 377 asparagine residue by glycine is responsible for the phenotype of the triple mutant. The effect of pH on the apparent hypoxanthine binding dissociation constant showed that the effects of N377G and N377I changes were, at least partially, due to a shift of the pKa of an ionizable amino acid residue of the unliganded permease. These two amino acid residue changes induced a shift of the pKa of this group in the unliganded, deprotonated permease about two units toward acidic pH. This result suggests that the 371-377 segment might play a key role in the proper three-dimensional structure of the active purine-cytosine permease.

In vivo phosphorylation of the purine/cytosine permease from the plasma membrane of the yeast Saccharomyces cerevisiae.

The purine/cytosine permease, encoded by the FCY2 gene, is a carrier located in the plasma membrane of the yeast Saccharomyces cerevisiae. Polyclonal antibodies were raised against two peptides that corresponded to the sub-N-terminal and C-terminal sequences of the putative protein deduced from the FCY2 gene. Immunoprecipitation experiments performed with protein extracts labelled in vivo with 35S showed that purine/cytosine permease is specifically detected as a broad and diffuse band. The apparent molecular mass of this protein was 45-50 kDa. By means of in vivo pulse/chase 35S-labelling experiments, we observed a slight increase in the apparent molecular mass of purine/cytosine permease during the chase. This shift in electrophoretic mobility of the protein suggested a post-translational modification. This molecular mass increase was eliminated by alkaline phosphatase treatment of the immunoprecipitate, which strongly suggested phosphorylation of the carrier. This proposal was confirmed by in vivo [32P]P(i) labelling and immunoprecipitation of purine/cytosine permease with purified anti-(sub-N-terminal peptide) IgG or anti-(C-terminal peptide) IgG. Phosphoamino acid analysis indicated that phosphorylation occurred on seryl residues of purine/cytosine permease. By means of thermosensitive secretory-pathway-mutant strains, we demonstrated that purine/cytosine permease phosphorylation occurred either between the Golgi apparatus and the plasma membrane or in the plasma membrane itself.

Purine-cytosine permease of Saccharomyces cerevisiae. Effect of external pH on nucleobase uptake and binding.

The cloned FCY2 gene (strain pAB4) of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the cloned allele fcy2-21 (strain pAB25) introduced into an S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus [Weber, E., Rodriguez, C., Chevallier, M. R. & Jund, R. (1990) Mol. Microbiol. 4, 585-596] were studied. The influence of external pH (varying over 3.5-6) has been analysed on the uptake of adenine, hypoxanthine and cytosine (Ktapp, apparent Michaelis constant and Vm) and on the binding constants of these three solutes (Kdapp, apparent half-saturation constant and Bmax, total binding sites) determined on plasma membranes. For pAB4, the variations of Ktapp and Vm were the same for the three bases, i.e. an increase in Ktapp when the pH increased and a maximum Vm around pH 5. For pAB25, Ktapp values varied in the same way and were significantly higher for the three bases than those found in pAB4. There was almost no variation of Vm for adenine, and there was a continuous decrease when the pH increased in the Vm of hypoxanthine and cytosine. Equilibrium binding measurements were performed for the three bases with plasma membrane isolated from pAB4 and pAB25. One single class of binding sites was detected. For pAB4, the affinity increased when the pH decreased for the three bases. The affinity of PCP for adenine was always greater than for cytosine or hypoxanthine. For pAB25, the same phenomenon was observed. However, the curves showing the variation of Kdapp as a function of pH were shifted towards more acidic pH values. A model was used to fit the experimental binding data obtained with hypoxanthine for the calculation of the dissociation constants of its binding to PCP and to determine the ionization constants of an amino acid involved in ligand binding. For pAB4, at acid pH, the dissociation constant was 1.7 +/- 0.4 microM. An amino acid displaying a pK of 3.8 was determined; this value was shifted to pK 4.8 when hypoxanthine was bound. For pAB25, the main effects of the mutation were a large decrease in the affinity of PCP for hypoxanthine (Kd of 14.4 +/- 4.3 microM) and a shift in the pK of the amino acid towards a more acidic pH (about 2.9). The pK of this group remained similar to the value obtained with pAB4 when hypoxanthine was bound. From these data, it is proposed that the binding of hypoxanthine and H+ is a random process.

In vivo and in vitro studies of the purine-cytosine permease of Saccharomyces cerevisiae. Functional analysis of a mutant with an altered apparent transport constant of uptake.

The FCY2 gene of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2-21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2-21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Ktapp) and Vmax] and on plasma-membrane preparations, by measurements of binding parameters at equilibrium [(Kd and maximum amount of binding sites/Bmax)]. For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered [1.8-2.6 microM and 8.5-10.2 nmol.min-1.(10(7) cells)-1]. The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine. Plasma membranes of each strain displayed one class of specific binding sites. Variations in Kd of 0.4-1 microM were observed for pAB4. These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 microM, 40 microM and 96 microM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine. Therefore, we conclude that modification in the Ktapp of uptake in the strain carrying fcy2-21 allele is merely due to a modification of the binding ability of the permease for its ligands.

Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae.

8-Azidoadenine was used as a photoaffinity reagent to characterize the purine-cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells. In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4-5-fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine-cytosine permease.

Quantitative determination of the calcium involved in the regulation of the Ca2+-ATPase activity in sarcoplasmic reticulum vesicles.

The dependence of the Ca2+-ATPase activity of sarcoplasmic reticulum vesicles upon the intravesicular concentration of calcium accumulated after active uptake was studied. The internal calcium concentration was modified by addition of the ionophore A23187 at the steady state of accumulation. About half of the calcium accumulated could be released at low ionophore concentration without any concomitant activation of the Ca2+-ATPase. This population of calcium might consist of calcium free in the lumen of the vesicles or bound to the bilayer at sites which do not interact with the ATPase activity. At higher concentrations of ionophore (above 1.75 nmol A23187/mg protein) the release of calcium activated this enzyme. This phenomenon was independent of the extravesicular calcium concentration and might be explained by assuming second species of calcium ions bound to the inner side of the membrane and in close functional interaction with the Ca2+-ATPase.

Depolarization-induced Ca2+ increase in isolated neurosecretory nerve terminals measured with fura-2.

The free Ca2+ concentration in isolated rat neurohypophysial nerve endings was measured using the Ca2+ indicator fura-2. Depolarization with high K, veratridine, or electrical stimulation induced an increase in intracellular Ca2+ concentration that was abolished by agents known to block voltage-sensitive Ca channels. Electrical stimulation of the isolated nerve endings with a pulse pattern similar to that recorded in vivo from the hypothalamic magnocellular neurons showed that the increase in intracellular Ca2+ concentration was not only a function of the applied frequency but also of the duration of the silent interburst intervals. The relationship between the cytoplasmic free Ca concentration and the release of neuropeptides is discussed.