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Abstract: FLR1 gene, encoding a multidrug resistance (MDR) transporter of the major facilitator superfamily (MFS) was found to confer resistance to the fungicide mancozeb in Saccharomyces cerevisiae. This agrochemical has been linked to the development of Parkinson disease and cancer. Yeast response to mancozeb was proved to involve the strong activation of FLR1 transcription (20-fold) during the fungicide-induced growth latency. This activation of FLR1 transcription is fully dependent on Yap1p and is reduced (by 50%) in the absence of Rpn4p, Yrr1p or Pdr3p. A model for the coordinate action over FLR1 transcription activation, in response to mancozeb, of these transcription factors that mediate oxidative stress response (Yap1p), proteasome gene expression (Rpn4p), and pleiotropic drug resistance (Pdr3p and Yrr1p), is proposed. [Copyright &y& Elsevier]

The pro-oxidant action of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is demonstrated in this study using Saccharomyces cerevisiae as a eukaryotic experimental model. Evidence is presented for the generation of hydroxyl-radicals, in yeast cells suddenly exposed to 2,4-D, detected by in vivo electron paramagnetic resonance (EPR) spectroscopy using 5,5′-dimethyl-1-pyrroline N-oxide and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide as spin-traps. The intensity of the EPR spectra was dependent on the concentration of herbicide tested and was consistently higher in a mutant (Δsod1) devoid of the cytosolic CuZn-superoxide dismutase. A time-course-dependent variation of the level of free-radical adducts was registered upon sudden exposure of an yeast cell population to concentrations of 2,4-D that lead to an initial period of viability loss, before resumption of inhibited growth by the viable adapted population. The variation pattern of the level of hydroxyl-radical adducts correlated with the one determined for the activity of Sod1p, cytosolic catalase Ctt1p, and the dithiol glutaredoxins Grx1p and Grx2p. [Copyright &y& Elsevier]

The transcription regulator Pdr1p is a determinant of Saccharomyces cerevisiae resistance to 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The Pdr1p-regulated genes, TPO1 and PDR5, encoding putative multidrug transporters belonging to the major facilitator superfamily (MFS) and to the ATP-binding cassette (ABC) superfamily, respectively, are required for yeast resistance to sudden exposure to these herbicides. A rapid and transient activation of TPO1 (sixfold) and PDR5 (twofold) transcription takes place during the adaptation period preceding cell division under MCPA or 2,4-D moderate stress. These activations are mediated by both Pdr1p and Pdr3p and, as soon as adapted cells start duplication under herbicide stress, mRNA levels are drastically reduced to basal values. The longer duration of the adaptation period, observed for the Δpdr1 population, may involve the abolishment of the Pdr1p-mediated transcriptional activation of TPO1 and PDR5 genes, whose expression is critical to surpass the viability loss during the initial period of adaptation to the herbicides. [Copyright &y& Elsevier]

Abstract: Succinate:quinone oxidoreductase (SQR), a di-haem enzyme purified from Rhodothermus marinus, reveals an HQNO-sensitive succinate:quinone oxidoreductase activity with several menaquinone analogues as electron acceptors that decreases with lowering the redox midpoint potential of the quinones. A turnover with the low-potential 2,3-dimethyl-1,4-naphthoquinone that is the closest analogue of menaquinone, although low, can be detected in liposome-reconstituted SQR. Reduction of the quinone is not stimulated by an imposed K+-diffusion membrane potential of a physiological sign (positive inside the vesicles). Nor does the imposed membrane potential increase the reduction level of the haems in R. marinus SQR poised with the succinate/fumarate redox couple. The data do not support a widely discussed hypothesis on the electrogenic transmembrane electron transfer from succinate to menaquinone catalysed by di-haem SQRs. The role of the membrane potential in regulation of the SQR activity is discussed. [Copyright &y& Elsevier]

Abstract: Yeast cells adapted to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) exhibit a plasma membrane less susceptible to 2,4-D-induced disruption and are more tolerant than unadapted cells to lethal concentrations of the herbicide. These cells, adapted to grow in the presence of increasing concentrations of 2,4-D, were found to exhibit a dose-dependent increase of the saturation degree of membrane fatty acids, associated to the higher percentage of stearic (C18:0) and palmitic (C16:0) acids, and to the decreased percentage of palmitoleic (Δ9-cisC16:1) and oleic (Δ9-cisC18:1) acids. The decreased transcription of the OLE1 gene (encoding the Δ9 fatty acid desaturase that catalyses the conversion of palmitic and stearic acids to palmitoleic and oleic acids, respectively) registered in 2,4-D adapted cells suggests that yeast adaptation to the herbicide involves the enhancement of the ratio of saturated (C16:0 and C18:0) to monounsaturated (C16:1 and C18:1) membrane fatty acids through a reduced OLE1 expression. [Copyright &y& Elsevier]

Abstract: Saccharomyces cerevisiae ORF YBR043c, predicted to code for a transporter of the major facilitator superfamily required for multiple drug resistance, encodes a plasma membrane protein that confers resistance to quinidine and barban, as observed before for its close homologues QDR1 and QDR2. This ORF was, thus, named the QDR3 gene. The increased expression of QDR3, or QDR2, also leads to increased resistance to the anticancer agents cisplatin and bleomycin. However, no evidence for increased QDR3 expression in yeast cells exposed to all these inhibitory compounds was found. Transport assays support the concept that Qdr3 is involved, even if opportunistically, in the active export of quinidine out of yeast cell. A correlation was established between the efficiency of quinidine active export mediated by Qdr3p, Qdr2p or Qdr1p, and the efficacy of the expression of the encoding genes in alleviating the deleterious action of quinidine, as well as of the other compounds (QDR2>QDR3>>>QDR1). [Copyright &y& Elsevier]

Abstract: In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo 10–nqo 14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE. [Copyright &y& Elsevier]

Abstract: Archaeoglobus fulgidus neelaredoxin (Nlr) is an electron donor:superoxide oxidoreductase. The reaction of superoxide with reduced Nlr is almost diffusion-limited, but the overall efficiency for detoxifying superoxide in vivo depends on the rate of reduction of Nlr by electron donors. Here, we report the purification and characterization of the two type I rubredoxins from A. fulgidus (AF0880 and AF1349) and show that they act as efficient electron donors for neelaredoxin, in vitro, with a second-order rate constant of 107M−1s−1 at 10°C and pH 7.2. [Copyright &y& Elsevier]

The A-type flavoproteins (ATF) are modular proteins involved in multi-component electron transfer pathways, having oxygen reductase activity. They are complex flavoproteins containing two distinct structural domains, one having an FMN in a flavodoxin-like fold and the other a binuclear iron centre within a metallo-β-lactamase-like fold. Here, we report the purification and characterisation of a recombinant ATF from the cyanobacterium Synechoystis sp. PCC 6803, which has the unique feature of comprising an additional third domain with similarities towards flavin:NAD(P)H reductases. The latter was expressed independently as a truncated protein form and found to be capable of receiving electrons from NADH as well as to indiscriminately bind either one FAD or one FMN with equivalent affinities. Further kinetic studies have shown that the intact ATF is an NADH:oxygen oxidoreductase, with the catalytic ability to fully reduce oxygen to water. Thus, this constitutes an example on how structural modules found within partner proteins from an electron transfer pathway can be combined in a single polypeptide chain achieving identical catalytic activities. [Copyright &y& Elsevier]

We report results on the functional analysis of Saccharomyces cerevisiae ORF YNL065w, predicted to code for a protein belonging to the poorly characterized major facilitator superfamily (MFS) of transporters that are involved in multidrug resistance (MDR). YNL065w is important for a moderate increase of yeast tolerance to ketoconazole and to the cationic dye crystal violet; it protects the cell against short-chain monocarboxylic acids (C2–C6), but not against highly liposoluble acids such as octanoic acid or the phenoxyacetic-acid herbicides 2,4-D and MCPA; it is also a determinant of resistance to the antiarrhytmic and antimalarial drug quinidine. The encoding ORF was, thus, denominated the AQR1 gene. Results obtained using an AQR1-lacZ fusion indicate that gene expression is very low and it is not stimulated under weak acid stress. The encoded putative transporter was localized in the plasma membrane by fluorescence microscopy observation of the overproduced Aqr1-GFP fusion protein distribution. [Copyright &y& Elsevier]