Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase.

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

DNA binding specificities of Escherichia coli Cas1-Cas2 integrase drive its recruitment at the CRISPR locus.

Prokaryotic adaptive immunity relies on the capture of fragments of invader DNA (protospacers) followed by their recombination at a dedicated acceptor DNA locus. This integrative mechanism, called adaptation, needs both Cas1 and Cas2 proteins. Here, we studied in vitro the binding of an Escherichia coli Cas1-Cas2 complex to various protospacer and acceptor DNA molecules. We show that, to form a long-lived ternary complex containing Cas1-Cas2, the acceptor DNA must carry a CRISPR locus, and the protospacer must not contain 3΄-single-stranded overhangs longer than 5 bases. In addition, the acceptor DNA must be supercoiled. Formation of the ternary complex is synergistic, in such that the binding of Cas1-Cas2 to acceptor DNA is reinforced in the presence of a protospacer. Mutagenesis analysis at the CRISPR locus indicates that the presence in the acceptor plasmid of the palindromic motif found in CRISPR repeats drives stable ternary complex formation. Most of the mutations in this motif are deleterious even if they do not prevent cruciform structure formation. The leader sequence of the CRISPR locus is fully dispensable. These DNA binding specificities of the Cas1-Cas2 integrase are likely to play a major role in the recruitment of this enzyme at the CRISPR locus.

Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae.

Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity.

RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase.

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.

Uptake of selenite by Saccharomyces cerevisiae involves the high and low affinity orthophosphate transporters.

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P(i) concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P(i) transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P(i) medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P(i) transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.

Structure of an archaeal heterotrimeric initiation factor 2 reveals a nucleotide state between the GTP and the GDP states.

Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/aIF)1 and the heterotrimeric initiation factor e/aIF2. In its GTP-bound form, e/aIF2 provides the initiation complex with Met-tRNA(i)(Met). After recognition of the start codon by initiator tRNA, e/aIF1 leaves the complex. Finally, e/aIF2, now in a GDP-bound form, loses affinity for Met-tRNA(i)(Met) and dissociates from the ribosome. Here, we report a 3D structure of an aIF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit gamma exchange conformational information with alpha and beta. The zinc-binding domain of beta lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the "ON" conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDP-inorganic phosphate (Pi) bound state of aIF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of eIF2 from the initiation complex is closely coupled to that of Pi from eIF2gamma upon start codon recognition. The nucleotide state of aIF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/aIF2gamma has been informed of e/aIF1 dissociation by e/aIF2beta.

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes.

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.

GEK1, a gene product of Arabidopsis thaliana involved in ethanol tolerance, is a D-aminoacyl-tRNA deacylase.

GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal d-Tyr-tRNA(Tyr) deacylases. To determine whether it actually possessed deacylase activity, the product of the GEK1 open reading frame was expressed in Escherichia coli from a multi-copy plasmid. Purified GEK1 protein contains two zinc ions and proves to be a broad-specific, markedly active d-aminoacyl-tRNA deacylase in vitro. Moreover, GEK1 expression is capable of functionally compensating in E. coli for the absence of endogeneous d-Tyr- tRNA(Tyr) deacylase. Possible connections between exposure of plants to ethanol/acetaldehyde and misaminoacylation of tRNA by d-amino acids are considered.

Structural switch of the gamma subunit in an archaeal aIF2 alpha gamma heterodimer.

Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (alphabetagamma) supplying the small subunit of the ribosome with methionylated initiator tRNA. This study reports the crystallographic structure of an aIF2alphagamma heterodimer from Sulfolobus solfataricus bound to Gpp(NH)p-Mg(2+). aIF2gamma is in a closed conformation with the G domain packed on domains II and III. The C-terminal domain of aIF2alpha interacts with domain II of aIF2gamma. Conformations of the two switch regions involved in GTP binding are similar to those encountered in an EF1A:GTP:Phe-tRNA(Phe) complex. Comparison with the EF1A structure suggests that only the gamma subunit of the aIF2alphagamma heterodimer contacts tRNA. Because the alpha subunit markedly reinforces the affinity of tRNA for the gamma subunit, a contribution of the alpha subunit to the switch movements observed in the gamma structure is considered.

Protection-based assays to measure aminoacyl-tRNA binding to translation initiation factors.

To decipher the mechanisms of translation initiation, the stability of the complexes between tRNA and initiation factors has to be evaluated in a routine manner. A convenient method to measure the parameters of binding of an aminoacyl-tRNA to an initiation factor results from the property that, when specifically complexed to a protein, the aminoacyl-tRNA often resists spontaneous deacylation. This chapter describes the preparation of suitable aminoacyl-tRNA ligands and their use in evaluating the stability of their complexes with various initiation factors, such as e/aIF2 and e/aIF5B. The advantages and the limitations of the method are discussed.

Structural basis for tRNA-dependent amidotransferase function.

Besides direct charging of tRNAs by aminoacyl-tRNA synthetases, indirect routes also ensure attachment of some amino acids onto tRNA. Such routes may explain how new amino acids entered into protein synthesis. In archaea and in most bacteria, tRNA(Gln) is first misaminoacylated by glutamyl-tRNA synthetase. Glu-tRNA(Gln) is then matured into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report the structure of a tRNA-dependent amidotransferase-that of GatDE from Pyrococcus abyssi. The 3.0 A resolution crystal structure shows a tetramer with two GatD molecules as the core and two GatE molecules at the periphery. The fold of GatE cannot be related to that of any tRNA binding enzyme. The ammonium donor site on GatD and the tRNA site on GatE are markedly distant. Comparison of GatD and L-asparaginase structures shows how the motion of a beta hairpin region containing a crucial catalytic threonine may control the overall reaction cycle of GatDE.

Recent advances in the mechanism of selenoamino acids toxicity in eukaryotic cells.

Selenium is an essential trace element due to its incorporation into selenoproteins with important biological functions. However, at high doses it is toxic. Selenium toxicity is generally attributed to the induction of oxidative stress. However, it has become apparent that the mode of action of seleno-compounds varies, depending on its chemical form and speciation. Recent studies in various eukaryotic systems, in particular the model organism Saccharomyces cerevisiae, provide new insights on the cytotoxic mechanisms of selenomethionine and selenocysteine. This review first summarizes current knowledge on reactive oxygen species (ROS)-induced genotoxicity of inorganic selenium species. Then, we discuss recent advances on our understanding of the molecular mechanisms of selenocysteine and selenomethionine cytotoxicity. We present evidences indicating that both oxidative stress and ROS-independent mechanisms contribute to selenoamino acids cytotoxicity. These latter mechanisms include disruption of protein homeostasis by selenocysteine misincorporation in proteins and/or reaction of selenols with protein thiols.

Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae.

Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress. We demonstrate that selenomethionine induces an accumulation of protein aggregates by a mechanism that requires de novo protein synthesis. Reduction of translation rates was accompanied by a decrease of protein aggregation and of selenomethionine toxicity. Protein aggregation was supressed in a ∆cys3 mutant unable to synthetize selenocysteine, suggesting that aggregation results from the metabolization of selenomethionine to selenocysteine followed by translational incorporation in the place of cysteine. In support of this mechanism, we were able to detect random substitutions of cysteinyl residues by selenocysteine in a reporter protein. Our results reveal a novel mechanism of toxicity that may have implications in higher eukaryotes.

Peptidyl-tRNA hydrolase from Sulfolobus solfataricus.

An enzyme capable of liberating functional tRNA(Lys) from Escherichia coli diacetyl-lysyl-tRNA(Lys) was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA(fMet) as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNA(Lys) from diacetyl-lysyl-tRNA(Lys). Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.

Use of analogues of methionine and methionyl adenylate to sample conformational changes during catalysis in Escherichia coli methionyl-tRNA synthetase.

Binding of methionine to methionyl-tRNA synthetase (MetRS) is known to promote conformational changes within the active site. However, the contribution of these rearrangements to enzyme catalysis is not fully understood. In this study, several methionine and methionyl adenylate analogues were diffused into crystals of the monomeric form of Escherichia coli methionyl-tRNA synthetase. The structures of the corresponding complexes were solved at resolutions below 1.9A and compared to those of the enzyme free or complexed with methionine. Residues Y15 and W253 play key roles in the strength of the binding of the amino acid and of its analogues. Indeed, full motions of these residues are required to recover the maximum in free energy of binding. Residue Y15 also controls the size of the hydrophobic pocket where the amino acid side-chain interacts. H301 appears to participate to the specific recognition of the sulphur atom of methionine. Complexes with methionyl adenylate analogues illustrate the shielding by MetRS of the region joining the methionine and adenosine moieties. Finally, the structure of MetRS complexed to a methionine analogue mimicking the tetrahedral carbon of the transition state in the aminoacylation reaction was solved. On the basis of this model, we propose that, in response to the binding of the 3'-end of tRNA, Y15 moves again in order to deshield the anhydride bond in the natural adenylate.

Neutralization by metal ions of the toxicity of sodium selenide.

Inert metal-selenide colloids are found in animals. They are believed to afford cross-protection against the toxicities of both metals and selenocompounds. Here, the toxicities of metal salt and sodium selenide mixtures were systematically studied using the death rate of Saccharomyces cerevisiae cells as an indicator. In parallel, the abilities of these mixtures to produce colloids were assessed. Studied metal cations could be classified in three groups: (i) metal ions that protect cells against selenium toxicity and form insoluble colloids with selenide (Ag⁺, Cd²âº, Cu²âº, Hg²âº, Pb²âº and Zn²âº), (ii) metal ions which protect cells by producing insoluble metal-selenide complexes and by catalyzing hydrogen selenide oxidation in the presence of dioxygen (Co²âº and Ni²âº) and, finally, (iii) metal ions which do not afford protection and do not interact (Ca²âº, Mg²âº, Mn²âº) or weakly interact (Fe²âº) with selenide under the assayed conditions. When occurring, the insoluble complexes formed from divalent metal ions and selenide contained equimolar amounts of metal and selenium atoms. With the monovalent silver ion, the complex contained two silver atoms per selenium atom. Next, because selenides are compounds prone to oxidation, the stabilities of the above colloids were evaluated under oxidizing conditions. 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the reduction of which can be optically followed, was used to promote selenide oxidation. Complexes with cadmium, copper, lead, mercury or silver resisted dissolution by DTNB treatment over several hours. With nickel and cobalt, partial oxidation by DTNB occurred. On the other hand, when starting from ZnSe or FeSe complexes, full decompositions were obtained within a few tens of minutes. The above properties possibly explain why ZnSe and FeSe nanoparticles were not detected in animals exposed to selenocompounds.

Sodium selenide toxicity is mediated by O2-dependent DNA breaks.

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (•)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.

Translation Initiation.

Selection of correct start codons on messenger RNAs is a key step required for faithful translation of the genetic message. Such a selection occurs in a complex process, during which a translation-competent ribosome assembles, eventually having in its P site a specialized methionyl-tRNAMet base-paired with the start codon on the mRNA. This chapter summarizes recent advances describing at the molecular level the successive steps involved in the process. Special emphasis is put on the roles of the three initiation factors and of the initiator tRNA, which are crucial for the efficiency and the specificity of the process. In particular, structural analyses concerning complexes containing ribosomal subunits, as well as detailed kinetic studies, have shed new light on the sequence of events leading to faithful initiation of protein synthesis in Bacteria.

NMR-based substrate analog docking to Escherichia coli peptidyl-tRNA hydrolase.

Escherichia coli peptidyl-tRNA hydrolase activity is inhibited by 3'-(L-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine, a stable mimic of the minimalist substrate 2'(3')-O-(L-[N,N-diacetyl-lysinyl)adenosine. The complex of this mimic with the enzyme has been analyzed by NMR spectroscopy, enabling experimental mapping of the catalytic center for the first time. Chemical shift variations point out the sensitivity of residues Asn10, Met67, Asn68, Gly111, Asn114, Leu116, Lys117, Gly147, Phe148, and Val149 to complex formation. Docking simulations based on ambiguous interaction restraints involving these residues show bondings of the peptide moiety of 3'-(l-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine with Asn10, Asn68, and Asn114. A stacking interaction of Phe66 with the purine is also indicated. Drawn is a model of enzyme-bound peptidyl-tRNA substrate, in which: (i) the Asn114 δ(2) NH(2) group holds the water molecule that participates in the hydrolysis of the substrate, while Tyr15 binds the phosphate in the 5'-position of the 3'-terminal tRNA adenosine; (ii) the δ(2) NH(2) group of Asn68 holds the main-chain carbonyl of the C-terminal residue of the peptide esterified to tRNA; and (iii) the δ(2) NH(2) group of Asn10 holds the main-chain carbonyl of the penultimate C-residue. Functional value is given to this model by (i) showing that the enzyme becomes confusable with an aminoacyl-tRNA hydrolase upon mutagenesis of Asn10 and (ii) reinterpreting already obtained site-directed mutagenesis data.

Selenodiglutathione uptake by the Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p.

The Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p is involved in heavy metal detoxification by mediating the ATP-dependent transport of glutathione-metal conjugates to the vacuole. In the case of selenite toxicity, deletion of YCF1 was shown to confer increased resistance, rather than sensitivity, to selenite exposure [Pinson B, Sagot I & Daignan-Fornier B (2000) Mol Microbiol36, 679-687]. Here, we show that when Ycf1p is expressed from a multicopy plasmid, the toxicity of selenite is exacerbated. Using secretory vesicles isolated from a sec6-4 mutant transformed either with the plasmid harbouring YCF1 or the control plasmid, we establish that the glutathione-conjugate selenodigluthatione is a high-affinity substrate of this ATP-binding cassette transporter and that oxidized glutathione is also efficiently transported. Finally, we show that the presence of Ycf1p impairs the glutathione/oxidized glutathione ratio of cells subjected to a selenite stress. Possible mechanisms by which Ycf1p-mediated vacuolar uptake of selenodiglutathione and oxidized glutathione enhances selenite toxicity are discussed.