A highly specific phosphatase that acts on ADP-ribose 1''-phosphate, a metabolite of tRNA splicing in Saccharomyces cerevisiae

One molecule of ADP-ribose 1″,2″-cyclic phosphate (Appr>p) is formed during each of the approximately 500000 tRNA splicing events per Saccharomyces cerevisiae generation. The metabolism of Appr>p remains poorly defined. A cyclic phosphodiesterase (Cpd1p) has been shown to convert Appr>p to ADP-ribose-1″-phosphate (Appr1p). We used a biochemical genomics approach to identify two yeast phosphatases that can convert Appr1p to ADP-ribose: the product of ORF YBR022w (now Poa1p), which is completely unrelated to other known phosphatases; and Hal2p, a known 3′-phosphatase of 5′,3′-pAp. Poa1p is highly specific for Appr1p, and thus likely acts on this molecule in vivo. Poa1 has a relatively low K_M for Appr1p (2.8 μM) and a modest k_cat (1.7 min⁻¹), but no detectable activity on several other substrates. Furthermore, Poa1p is strongly inhibited by ADP-ribose (K_I, 17 μM), modestly inhibited by other nucleotides containing an ADP-ribose moiety and not inhibited at all by other tested molecules. In contrast, Hal2p is much more active on pAp than on Appr1p, and several other tested molecules were Hal2p substrates or inhibitors. poa1-Δ mutants have no obvious growth defect at different temperatures in rich media, and analysis of yeast extracts suggests that ~90% of Appr1p processing activity originates from Poa1p.     

Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase

The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake in Saccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is necessary for high-affinity potassium uptake even in the absence of Na⁺ stress. HAL5 expression is induced in response to stress in a calcineurin-dependent manner through a newly identified functional CDRE (nt −195/−189). Lack of calcineurin decreases Hal5 protein levels, although with little effect on Trk1 amounts. However, the growth defect of cnb1 cells at K⁺-limiting conditions can be rescued in part by overexpression of HAL5, and this mutation further aggravates the potassium requirements of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expression may be biologically relevant for Trk1 regulation.     

Expressing the yeast HAL1 gene in tomato increases fruit yield and enhances K+/Na+ selectivity under salt stress

The yeast HAL1 gene facilitates K⁺/Na⁺ selectivity and salt tolerance of cells. Ectopic expression of HAL1 in transgenic tomato (Lycopersicon esculentum Mill.) plants minimized the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K⁺ contents in both leaf calli and leaves in the presence of salt, which indicates that HAL1 functions in plants using a similar mechanism to that in yeast, namely by facilitating K⁺/Na⁺ selectivity under salt stress.     

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.     

Vacuolar Cation/H+ Antiporters of Saccharomyces cerevisiae

We previously demonstrated that Saccharomyces cerevisiae vnx1Δ mutant strains displayed an almost total loss of Na⁺ and K⁺/H⁺ antiporter activity in a vacuole-enriched fraction. However, using different in vitro transport conditions, we were able to reveal additional K⁺/H⁺ antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1Δvcx1Δ nhx1Δ triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca²⁺/H⁺ antiporter activity catalyzed by Vcx1p, the K⁺/H⁺ antiporter activity was strongly inhibited by Cd²⁺ and to a lesser extend by Zn²⁺. Unlike as previously observed for NHX1 or VNX1, VCX1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K⁺ and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1p and Vnx1p are targeted to the vacuolar membrane, whereas Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed.     

The uniqueness of the plant mitochondrial potassium channel

The ATP-inhibited Plant Mitochondrial K⁺ Channel (PmitoK_ATP) was discovered about fifteen years ago in Durum Wheat Mitochondria (DWM). PmitoKATP catalyses the electrophoretic K⁺ uniport through the inner mitochondrial membrane; moreover, the co-operation between PmitoK_ATP and ⁺/H⁺ antiporter allows such a great operation of a K⁺ cycle to collapse mitochondrial membrane potential (ΔΨ) and ΔpH, thus impairing protonmotive force (Δp). A possible physiological role of such ΔΨ control is the restriction of harmful reactive oxygen species (ROS) production under environmental/oxidative stress conditions. Interestingly, DWM lacking Δp were found to be nevertheless fully coupled and able to regularly accomplish ATP synthesis; this unexpected behaviour makes necessary to recast in some way the classical chemiosmotic model. In the whole, PmitoK_ATP may oppose to large scale ROS production by lowering ΔΨ under environmental/oxidative stress, but, when stress is moderate, this occurs without impairing ATP synthesis in a crucial moment for cell and mitochondrial bioenergetics. [BMB Reports 2013; 46(8): 391-397]     

Transport of methionine and proline by rat liver slices and the effect of certain hormones

1. Transport characteristics of l-methionine and l-proline in rat liver slices in vitro were studied. 2. Intracellular concentration gradients for methionine were obtained. 3. Methionine uptake was inhibited by iodoacetate, dinitrophenol, Na⁺-free media and also by glycine, lysine, cysteine and dithiothreitol but not by α-aminoisobutyrate. 4. The rate of methionine metabolism in the slice was slow. 5. Puromycin inhibited methionine incorporation into protein, but not methionine uptake. 6. Methionine inhibited the transport of α-aminoisobutyrate but not of cystine. 7. Efflux and exchange diffusion of methionine was studied. 8. Amino acid transport in rat liver slices was not affected by thyroidectomy. 9. Addition of insulin, glucagon, adrenaline or cortisol did not affect the transport of methionine. 10. Addition of 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate increased methionine transport after a 120min incubation period in some experiments. 11. Studies of l-proline transport were invalidated because of the rapid evolution of CO₂ from the substrate.     

Broad sensitivity of Saccharomyces cerevisiae lacking ribosome-associated chaperone ssb or zuo1 to cations, including aminoglycosides

The Hsp70 Ssb and J protein Zuo1 of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, proposed to be involved in the folding of newly synthesized polypeptide chains. Cells lacking Ssb and/or Zuo1 have been reported to be hypersensitive to cationic aminoglycoside protein synthesis inhibitors that affect translational fidelity and to NaCl. Since we found that Δssb1 Δssb2 (Δssb1,2), Δzuo1, and wild-type cells have very similar levels of translational misreading in the absence of aminoglycosides, we asked whether the sensitivities to aminoglycosides and NaCl represent a general increase in sensitivity to cations. We found that Δssb1,2 and Δzuo1 cells are hypersensitive to a wide range of cations. This broad sensitivity is similar to that of cells having lowered activity of major plasma membrane transporters, such as the major K⁺ transporters Trk1 and Trk2 or their regulators Hal4 and Hal5. Like Δhal4,5 cells, Δssb1,2 and Δzuo1 cells have increased intracellular levels of Na⁺ and Li⁺ upon challenge with higher-than-normal levels of these cations, due to an increased rate of influx. In the presence of aminoglycosides, Δssb1,2, Δzuo1, and Δhal 4,5 cells have similarly increased levels of translational misreading. We conclude that, in vivo, the major cause of the aminoglycoside sensitivity of cells lacking ribosome-associated molecular chaperones is a general increase in cation influx, perhaps due to altered maturation of membrane proteins.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5′ to 3′ DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5′ to 3′ exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5′ to 3′ helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.Key words: yeast, RAD27, RAD9, RAD53, Okazaki fragment processing, DNA replication, exo1     

Dinucleotide Sequences at the 5' Ends of Vaccinia Virus mRNA's Synthesized In Vitro

The diversity of dinucleotide sequences at the 5′ ends of vaccinia virus mRNA's was determined by a two-dimensional electrophoresis procedure. RNA labeled with S-adenosyl[methyl-³H]methionine was synthesized in vitro by enzymes present in vaccinia virus cores. The RNA, ending in m⁷G(5′)pppN^mpN−, was β-eliminated and treated with alkaline phosphatase. After digestion with RNases T₂, T₁, and A, all eight possible dinucleotides containing G^m and A^m were identified. They are, in decreasing order of abundance: G^mpUp (22%), A^mpCp (18%), G^mpAp (16%), G^mpCp (15%), A^mpAp (11%), A^mpUp (10%), A^mpGp (7%), and G^mpGp (2%).     

In Vivo 23Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

Sodium gradients (ΔpNa) were measured in resting cells of Fibrobacter succinogenes by in vivo ²³Na nuclear magnetic resonance using Tm(DOTP)⁵⁻ [thulium(III) 1,4,7,10-tetraazacyclododecane-N′,N′′,N′′′-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a ΔpNa of −55 to −40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na⁺ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).     

Melting studies of dangling-ended DNA hairpins: effects of end length,loop sequence and biotinylation of loop bases

The effects of 3′ single-strand dangling-ends of different lengths, sequence identity of hairpin loop, and hairpin loop biotinylation at different loop residues on DNA hairpin thermodynamic stability were investigated. Hairpins contained 16 bp stem regions and five base loops formed from the sequence, 5′-TAGTCGACGTGGTCC-N₅-GGACCACGTCGACTAG-E_n-3′. The length of the 3′ dangling-ends (E_n) was n = 13 or 22 bases. The identities of loop bases at positions 2 and 4 were varied. Biotinylation was varied at loop base positions 2, 3 or 4. Melting buffers contained 25 or 115 mM Na⁺. Average t_m values for all molecules were 73.5 and 84.0°C in 25 and 115 mM Na⁺, respectively. Average two-state parameters evaluated from van’t Hoff analysis of the melting curve shapes in 25 mM Na⁺ were ΔH_vH = 84.8 ± 15.5 kcal/mol, ΔS_vH = 244.8 ± 45.0 cal/K·mol and ΔG_vH = 11.9 ± 2.1 kcal/mol. In 115 mM Na⁺, two-state parameters were not very different at ΔH_vH = 80.42 ± 12.74 kcal/mol, ΔS_vH = 225.24 ± 35.88 cal/K·mol and ΔG_vH = 13.3 ± 2.0 kcal/mol. Differential scanning calorimetry (DSC) was performed to test the validity of the two-state assumption and evaluated van’t Hoff parameters. Thermodynamic parameters from DSC measurements (within experimental error) agreed with van’t Hoff parameters, consistent with a two-state process. Overall, dangling-end DNA hairpin stabilities are not affected by dangling-end length, loop biotinylation or sequence and vary uniformly with [Na⁺]. Consider able freedom is afforded when designing DNA hairpins as probes in nucleic acid based detection assays, such as microarrays.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg²⁺ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li⁺ (50% inhibitory concentration [IC₅₀] = 3.6 mM) but only slightly sensitive to Na⁺ (IC₅₀ = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li⁺ and Na⁺, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li⁺ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

Bacillus subtilis polynucleotide phosphorylase 3′-to-5′ DNase activity is involved in DNA repair

In the presence of Mn²⁺, an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3′ → 5′ polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn²⁺ and low-level inorganic phosphate (P_i), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg²⁺ or high-level P_i. In contrast, the RNase activity of PNPase requires Mg²⁺ and P_i, suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (ΔpnpA) is not epistatic with ΔrecA, but is epistatic with ΔrecN and Δku, which by themselves are non-epistatic. The addA5, ΔrecO, ΔrecQ (ΔrecJ), ΔrecU and ΔrecG mutations (representative of different epistatic groups), in the context of ΔpnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.     

Light-Induced Proton Gradient Formation in Intact Cells of Dunaliella salina

A light-induced proton gradient (ΔpH) increase as exhibited by an increase of 9-aminoacridine fluorescence quenching is demonstrated between the external medium and the interior of the halophytic green alga Dunaliella salina. The formation and maintenance of the ΔpH is sensitive to electron transport inhibitors and to uncouplers. It is inhibited by p-chloromercuribenzenesulfonic acid (50% inhibition at 3 micromolar), which does not affect photosynthetic O₂ evolution. It is concluded that the observed ΔpH is located across the plasmalemma or the chloroplast envelope. The formation and maintenance of the light-induced proton gradient requires the presence of Na⁺. Substitution of NaCl by KCl or glycerol results in inhibition of the ΔpH formation. The proton gradient is also sensitive to ATPase and energy transfer inhibitors. It is suggested that a Na⁺/H⁺ pump mechanism may be involved in the formation of the proton gradient in intact Dunaliella cells.