Identification of critical residues for transport activity of Acr3p,the Saccharomyces cerevisiae As(III)/H+ antiporter

Acr3p is an As(III)/H⁺ antiporter from Saccharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that Cys151 located in the middle of the fourth transmembrane segment (TM4) is critical for antiport activity, suggesting that As(III) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in As(III)/H⁺ exchange, we performed a systematic alanine‐replacement analysis of charged/polar and aromatic residues that are conserved in the Acr3 family and located in putative transmembrane segments. Nine residues (Asn117, Trp130, Arg150, Trp158, Asn176, Arg230, Tyr290, Phe345, Asn351) were found to be critical for proper folding and trafficking of Acr3p to the plasma membrane. In addition, we found that replacement of highly conserved Phe266 (TM7), Phe352 (TM9), Glu353 (TM9) and Glu380 (TM10) with Ala abolished transport activity of Acr3p, while mutation of Ser349 (TM9) to Ala significantly reduced the As(III)/H⁺ exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of Glu353 and Glu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM9 and TM10 are crucial for antiport activity. We also discuss a hypothetical model of the Acr3p transport mechanism.     

Efflux permease CgAcr3-1 of Corynebacterium glutamicum is an arsenite-specific antiporter

Resistance to arsenite (As(III)) by cells is generally accomplished by arsenite efflux permeases from Acr3 or ArsB unrelated families. We analyzed the function of three Acr3 proteins from Corynebacterium glutamicum, CgAcr3-1, CgAcr3-2, and CgAcr3-3. CgAcr3-1 conferred the highest level of As(III) resistance and accumulation in vivo. CgAcr3-1 was also the most active when everted membranes vesicles from Escherichia coli or C. glutamicum mutants were assayed for efflux with different energy sources. As(III) and antimonite (Sb(III)) resistance and accumulation studies using E. coli or C. glutamicum arsenite permease mutants clearly show that CgAcr3-1 is specific for As(III). In everted membrane vesicles expressing CgAcr3-1, dissipation of either the membrane potential or the pH gradient of the proton motive force did not prevent As(III) uptake, whereas dissipation of both components eliminated uptake. Further, a mutagenesis study of CgAcr3-1 suggested that a conserved cysteine and glutamate are involved in active transport. Therefore, we propose that CgAcr3-1 is an antiporter that catalyzes arsenite-proton exchange with residues Cys129 and Glu305 involved in efflux.     

Functioning of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Pma1 H<Superscript>+</Superscript>-ATPase carrying the minimal number of cysteine residues

Pma1 H⁺-ATPase is the primary proton pump in the plasma membrane of the yeast Saccharomyces cerevisiae. It generates an electrochemical proton gradient, thus providing energy for secondary solute transport systems. The enzyme contains nine cysteines, three (Cys148, Cys312, and Cys867) in transmembrane segments and the rest (Cys221, Cys376, Cys409, Cys472, Cys532, and Cys569) in the cytosolic parts of the molecule. Although individually they are not essential for the functioning of the ATPase, substitution of all of them leads to the loss of enzyme activity and impairment of biogenesis. By means of site-directed mutagenesis combined with other molecular-biological and biochemical methods, this work defines different combinations of minimal cysteine content that are consistent with ATPase function.     

Acr3p is a plasma membrane antiporter that catalyzes As(III)/H(+) and Sb(III)/H(+) exchange in Saccharomyces cerevisiae

Resistance to arsenical compounds in Saccharomyces cerevisiae as well as in a growing number of prokaryotes and eukaryotes is mediated by members of the Acr3 family of transporters. In yeast cells, it has been clearly shown that Acr3p is localized to the plasma membrane and facilitates efflux of trivalent arsenic and antimony. However, until now, the energy dependence and kinetic properties of Acr3 proteins remained uncharacterized. In this work, we show that arsenite and antimonite uptake into everted membrane vesicles via the yeast Acr3 transporter is coupled to the electrochemical potential gradient of protons generated by the plasma membrane H(+)-translocating P-type ATPase. These results strongly indicate that Acr3p acts as a metalloid/H(+) antiporter. Two differential kinetic assays revealed that Acr3p-mediated arsenite/H(+) and antimonite/H(+) exchange demonstrates Michaelis-Menten-type saturation kinetics characterized by a maximum flux for permeating metalloids. The approximate K(m) values for arsenite and antimonite transport were the same, suggesting that Acr3p exhibits similar low affinity for both metalloids. Nevertheless, the maximal velocity of the transport at saturation concentrations of metalloids was approximately 3 times higher for arsenite than for antimonite. These findings may explain a predominant role of Acr3p in conferring arsenite tolerance in S. cerevisiae.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

Topological analyses of the L-lysine exporter LysO reveal a critical role for a conserved pair of intramembrane solvent-exposed acidic residues

LysO, a prototypical member of the LysO family, mediates export of L-lysine (Lys) and resistance to the toxic Lys antimetabolite, L-thialysine (Thl) in Escherichia coli. Here, we have addressed unknown aspects of LysO function pertaining to its membrane topology and the mechanism by which it mediates Lys/Thl export. Using substituted cysteine (Cys) accessibility, here we delineated the membrane topology of LysO. Our studies support a model in which both the N- and C-termini of LysO are present at the periplasmic face of the membrane with a transmembrane (TM) domain comprising eight TM segments (TMSs) between them. In addition, a feature of intramembrane solvent exposure in LysO is inferred with the identification of membrane-located solvent-exposed Cys residues. Isosteric substitutions of a pair of conserved acidic residues, one E233, located in the solvent-exposed TMS7 and the other D261, in a solvent-exposed intramembrane segment located between TMS7 and TMS8, abolished LysO function in vivo. Thl, but not Lys, elicited proton release in inside-out membrane vesicles, a process requiring the presence of both E233 and D261. We postulate that Thl may be exported in antiport with H⁺ and that Lys may be a low-affinity export substrate. Our findings are compatible with a physiological scenario wherein in vivo LysO exports the naturally occurring antimetabolite Thl with higher affinity over the essential cellular metabolite Lys, thus affording protection from Thl toxicity and limiting wasteful export of Lys.     

A novel method to quantify H-ATPase-dependent Na transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na⁺ is excluded from the cytosol to the apoplast or the vacuole by Na⁺/H⁺ antiporters. The secondary active transport of Na⁺ to apoplast against its electrochemical gradient is driven by plasma membrane H⁺-ATPases that hydrolyze ATP and pump H⁺ across the plasma membrane. Current methods to determine Na⁺ flux rely either on the use of Na-isotopes (²²Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H⁺ gradient due to H⁺-ATPase in the presence or absence of Na⁺ by pH-sensitive probes. To date, there are no methods that can directly quantify H⁺-ATPase-dependent Na⁺ transport in plasma membrane vesicles. We developed a method to measure bidirectional H⁺-ATPase-dependent Na⁺ transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H⁺-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na⁺ sealed for the study of H⁺-ATPase-dependent Na⁺ transport. Our data implicate that Na⁺ movement across vesicle membranes is highly dependent on H⁺-ATPase activity requiring ATP and Mg²⁺ and displays optimum rates of 2.50 μM Na⁺ mg^− 1 membrane protein min^− 1 at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H⁺-ATPase-dependent Na⁺ transport. The results demonstrate that the Na⁺ content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H⁺-ATPase-dependent Na⁺ transport with membrane vesicles.     

Electroneutral K/H exchange in mitochondrial membrane vesicles involves Yol027/Letm1 proteins

YOL027c in yeast and LETM1 in humans encode integral proteins of the inner mitochondrial membrane. They have been implicated in mitochondrial K⁺ homeostasis and volume control. To further characterize their role, we made use of submitochondrial particles (SMPs) with entrapped K⁺- and H⁺-sensitive fluorescent dyes PBFI and BCECF, respectively, to study the kinetics of K⁺ and H⁺ transport across the yeast inner mitochondrial membrane. Wild-type SMPs exhibited rapid, reciprocal translocations of K⁺ and H⁺ driven by concentration gradients of either of them. K⁺ and H⁺ translocations have stoichiometries similar to those mediated by the exogenous K⁺/H⁺ exchanger nigericin, and they are shown to be essentially electroneutral and obligatorily coupled. Moreover, [K⁺] gradients move H⁺ against its concentration gradient, and vice-versa. These features, as well as the sensitivity of K⁺ and H⁺ fluxes to quinine and Mg²⁺, qualify these activities as K⁺/H⁺ exchange reactions. Both activities are abolished when the yeast Yol027p protein is absent (yol027Δ mutant SMPs), indicating that it has an essential role in this reaction. The replacement of the yeast Yol027p by the human Letm1 protein restores K⁺/H⁺ exchange activity confirming functional homology of the yeast and human proteins. Considering their newly identified function, we propose to refer to the yeast YOL027c gene and the human LETM1 gene as yMKH1 and hMKH1, respectively.     

Importance of the seryl and threonyl residues of the fifth transmembrane domain to the substrate specificity of yeast plasma membrane Na+/H+ antiporters

The Zygosaccharomyces rouxii Na⁺/H⁺ antiporter Sod2-22p is a member of the subfamily of yeast plasma membrane Nha/ Sod antiporters that do not recognize potassium as their substrate. A functional study of two ZrSod2-22p mutated versions that improved the tolerance of a S. cerevisiae alkali-metal-cation sensitive strain to high extracellular concentration of KCl identified two polar non-charged amino-acid residues in the fifth transmembrane domain, Thr141 and Ser150, as being involved in substrate recognition and transport in yeast Nha/Sod antiporters. A reciprocal substitution of amino-acid residues with a hydroxyl group at these positions, T141S or S150T, produced a broadened cation selectivity of the antiporter for K⁺, in addition to Na⁺ and Li⁺. Site-directed mutagenesis of Ser150 showed that while the replacement of Ser150 with a small hydrophobic (valine) or negatively charged (aspartate) amino acid did not produce a significant change in ZrSod2-22p substrate specificity, the introduction of a positive charge at this position stopped the activity of the antiporter. This data demonstrates that the amino-acid composition of the fifth transmembrane domain, mainly the presence of amino acids containing hydroxyl groups in this part of the protein, is critical for the recognition and transport of substrates and could participate in conformational movements during the binding and/or cation transport cycle in yeast plasma membrane Na⁺/H⁺ antiporters.     

Characterization of the ion transport activity of the budding yeast Na/H antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.     

ArsD residues Cys12, Cys13, and Cys18 form an As(III)-binding site required for arsenic metallochaperone activity

The ArsA ATPase is the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. ArsD is a metallochaperone that delivers As(III) to ArsA, increasing its affinity for As(III), thus conferring resistance to environmental concentrations of arsenic. R773 ArsD is a homodimer with three vicinal cysteine pairs, Cys(12)-Cys(13), Cys(112)-Cys(113), and Cys(119)-Cys(120), in each subunit. Each vicinal pair binds As(III) or Sb(III). Alignment of the primary sequence of homologues of ArsD indicates that only the first vicinal cysteine pair, Cys(12)-Cys(13), and an additional cysteine, Cys(18), are conserved. The effect of cysteine-to-alanine substitutions and truncations were examined. By yeast two-hybrid analysis, nearly all of the ArsD mutants were able to interact with wild type ArsD, indicating that the mutations do not interfere with dimerization. ArsD mutants with alanines substituting for Cys(112), Cys(113), Cys(119), or Cys(120) individually or in pairs or truncations lacking the vicinal pairs retained ability to interact with ArsA and to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys(12), Cys(13), or Cys(18), individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. We propose that ArsD residues Cys(12), Cys(13), and Cys(18), but not Cys(112), Cys(113), Cys(119), or Cys(120), are required for delivery of As(III) to and activation of the ArsAB pump.     

Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III

Dipeptidyl peptidases III (DPPs III) form a distinct metallopeptidase family characterized by the unique HEXXGH motif. High susceptibility to inactivation by organomercurials suggests the presence of a reactive cysteine residue(s) in, or close to, their active site. Yeast DPP III contains five Cys, none of which is absolutely conserved within the family. In order to identify reactive residue(s), site-directed mutagenesis on yeast His₆-tagged DPP III was employed to substitute specifically all five cysteine residues to serine. The variant enzymes thus obtained were enzymatically active and showed an overall structure not greatly affected by the mutations as judged by circular dichroism. Analysis by native and SDS-PAGE under non-reducing conditions revealed the existence of a monomeric and dimeric form in all DPP III proteins except in the C130S, implying that dimerization of yeast DPP III is mediated by the surface-exposed cysteine 130.     

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Eukaryotic P-type plasma membrane H⁺-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H⁺-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H⁺-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.     

Arsenic methylation by a novel ArsM As(III) S‐adenosylmethionine methyltransferase that requires only two conserved cysteine residues

Arsenic (As) biomethylation is an important component of the As biogeochemical cycle that can influence As toxicity and mobility in the environment. Biomethylation of As is catalyzed by the enzyme arsenite (As[III]) S‐adenosylmethionine methyltransferase (ArsM). To date, all identified ArsM orthologs with As(III) methylation activities have four conserved cysteine residues, which are thought to be essential for As(III) methylation. Here, we isolated an As(III)‐methylating bacterium, Bacillus sp. CX‐1, and identified a gene encoding a S‐adenosylmethionine methyltranserase termed BlArsM with low sequence similarities (≤ 39%) to other ArsMs. BlArsM has six cysteine residues (Cys10, Cys11, Cys145, Cys193, Cys195 and Cys268), three of which (Cys10, Cys145 and Cys195) align with conserved cysteine residues found in most ArsMs. BlarsM is constitutively expressed in Bacillus sp. CX‐1. Heterologous expression of BlarsM conferred As(III) resistance. Purified BlArsM methylated both As(III) and methylarsenite (MAs[III]), with a final product of dimethylarsenate (DMAs[V]). When all six cysteines were individually altered to serine residues, only C145S and C195S derivatives lost the ability to methylate As(III) and MAs(III). The derivative C10S/C11S/C193S/C268S was still active. These results suggest that BlArsM is a novel As(III) S‐adenosylmethionine methyltransferase requiring only two conserved cysteine residues. A model of As(III) methylation by BlArsM is proposed.     

Triton X-100 inhibition of yeast plasma membrane associated NADH-dependent redox activities

Plasma membrane (PM) vesicles isolated from the yeast Saccharomyces cerevisiae (wild-type NCIM 3078, and a MG 21290 mutant pma 1-1) were used to monitor the effect of the detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (Chaps) and Triton X-100, on H⁺-ATPase (E.C. 3.6.1.35), NADH oxidase and NADH- hexacynoferrate (III)[HCF (III)] oxidoreductase (E.C. 1.6.99.3) activities. The results obtained show that Triton X-100 inhibited both membrane bound and solubilized NADH-dependent redox activities. The nature of this inhibition as determined for NADH–HCF(III) oxidoreductase was non-competitive and the Ki values for wild and mutant enzymes were 1.2?×?10^?5?M and 8.0?×?10^?6?M, respectively. The findings are interpreted, in view of the established reports, that the active site architecture of PM bound NADH-dependent oxidoreductase in yeast is likely to be different than in other eukaryotes.     

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.Arsenic is a carcinogen that ranks first on the Superfund List of Hazardous Substances (www.atsdr.cdc.gov). As a consequence of its environmental ubiquity, nearly every organism, from bacteria to humans, has genes that confer resistance to arsenic (1). The most common mechanism of arsenite resistance is efflux from cells catalyzed by members of three unrelated families of transporters. Homologues of the Mrp members of the ATP-binding cassette superfamily catalyze ATP-dependent pumping of As(III)-thiol complexes out of the cytosol. These include Mrp1 and Mrp2 in mammals that extrude As(GS)₃ into blood or bile (2), Ycf1p in yeast that extrudes As(GS)₃ into the vacuole (3), and PgpA in Leishmania that extrudes the As(III)-trypanothione complex into intracellular compartments (4). These pumps are generalized resistance pumps and are not specific for arsenite. In contrast, ArsB, the first identified member of the second family of arsenite efflux proteins, has the physiological role of conferring resistance to inorganic As(III) and Sb(III) (5, 6). The best characterized member of the ArsB family is that encoded by the arsRDABC operon of the conjugative R-factor R773 of Escherichia coli. ArsB is widespread in bacteria and archaea. It has 12 membrane-spanning segments (7), which is similar to members of the Major Facilitator Superfamily (8). It transports As(III) but has higher affinity for Sb(III). ArsB is an antiporter that catalyzes the exchange of trivalent metalloid for protons, coupling arsenite efflux to the electrochemical proton gradient (9).The third arsenic resistance transporter is Acr3, which is a member of the BART (bile/arsenite/riboflavin transporter) superfamily and includes members found in bacteria, archaea, and fungi and is more widely distributed than members of the ArsB family (10) (supplemental Fig. 1). Homologues have recently been identified in plant (Pteris vittata, NCBI accession number {"type":"entrez-protein","attrs":{"text":"ACN65413","term_id":"224814384","term_text":"ACN65413"}}ACN65413) and animal genomes (Danio rerio, NCBI accession number {"type":"entrez-protein","attrs":{"text":"XP_001921075","term_id":"189546053","term_text":"XP_001921075"}}XP_001921075). Unfortunately, the literature is confused by the fact that many members of the Acr3 family are annotated as ArsB even though they exhibit no significant sequence similarity to ArsB. The first identified member of this family is encoded by the ars operon of the skin (sigK intervening) element in the chromosome of Bacillus subtilis (11). The membrane topology of the B. subtilis Acr3 was recently investigated using translational fusions, but the results could not distinguish between 8 and 10 transmembrane-spanning segments (TMs)² (12). Fungal members of this family include the Saccharomyces cerevisiae Acr3p metalloid efflux protein (3, 13). Interestingly, yeast Acr3p appears to be selective for As(III) over Sb(III), which is surprising considering the similarity in chemical properties between the two metalloids. The properties of a more distant homologue from Shewanella oneidensis was examined recently (14). The S. oneidensis homologue confers resistance to arsenate but not arsenite. Similarly, the purified protein binds arsenate, not arsenite, indicating that this protein is not an Acr3 orthologue.Here we examined the properties of Acr3 orthologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum (supplemental Fig. 1). A. metalliredigens is a borate-tolerant Gram-positive alkaliphile and strict anaerobe that uses reduction of metals as electron acceptors (15). It is a novel metal-reducing bacterium that is distantly related to other commonly studied iron-reducing microorganisms. The genome of A. metalliredigens QYMF (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_009633","term_id":"150387853","term_text":"NC_009633"}}NC_009633) contains two novel ars operons, arsR₁B_acr3–1D₁A_1–1A_1–2 and arsR₂CB_acr3–2D₂A_2–1A_2–2. The two genes for the AmAcr3s were designated ars_acr3 because they are both in ars operons and are controlled by ArsR repressors, even though they are not homologues of ArsB. Interestingly, both ars operons have genes for ArsD and two genes corresponding to the two homologous halves of ArsA, which we designate AmArsA1 and AmArsA2. ArsD is an arsenic chaperone that transfers As(III) to ArsA (16), which then interacts with ArsB to extrude As(III) from the cells in an ATP-dependent manner (6, 17, 18). Whether or how Acr3 can replace ArsB in this process is a question of considerable interest.C. glutamicum is a Gram-positive soil bacterium that is used for commercial production of glutamate, lysine, and other amino acids, nucleotides, and vitamins and from which the genome sequence has been described (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_006958","term_id":"62388892","term_text":"NC_006958"}}NC_006958). It is highly arsenic-resistant and has three genes encoding Acr3 homologues (19). Two of the homologues are in ars operons regulated by ArsRs (arsR₁B_acr3–1C₁C_1′ and arsR₂B_acr3–2arsC₂) and a third orphan gene (arsB_acr3–3) that is not in an operon and may not be expressed to the same extent as the other two. (Again, the genes were misnamed arsB even though they encode Acr3 homologues.)The genes for AmAcr3 and CgAcr3 from the ars1 operons of the respective species were cloned and expressed in the arsenite-hypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon had been deleted (20). Both conferred resistance to arsenite but not arsenate or antimonite. Examination of the sequence of Acr3 homologues from many species indicates that there is conserved cysteine residues, Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 (supplemental Fig. 1). Those and other nonconserved cysteine residues were changed by mutagenesis, and substitution of only Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 led to loss of function, suggesting that the conserved cysteine residue participates in As(III) transport. A scanning cysteine accessibility method (SCAM) (21) was used to determine the transmembrane topology of AmAcr3. SCAM analysis is preferable to the use of gene fusions because there are minimal structural changes in the membrane protein, and the sidedness of inserted cysteines can be unambiguously determined with maleimide reagents of differing membrane permeability. A series of single cysteine mutants of AmAcr3 was constructed and the reactivity of each cysteine residue assayed. The results unambiguously demonstrate that Acr3 has 10 TMs.     

A Novel Plant Vacuolar Na+/H+ Antiporter Gene Evolved by DNA Shuffling Confers Improved Salt Tolerance in Yeast

Plant vacuolar Na⁺/H⁺ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na⁺ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance; however the relatively low V_max of the Na⁺/H⁺ exchange of the Na⁺/H⁺ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na⁺/H⁺ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na⁺/H⁺ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl, and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na⁺/H⁺ exchange activity and a slightly improved K⁺/H⁺ exchange activity.