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- Bienert, Gern, 2008, et al.
(author)
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A subgroup of plant aquaporins facilitate the bi-directional diffusion of As(OH)3 and Sb(OH)3 across membranes
- 2008
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In: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 6:26
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Journal article (peer-reviewed)abstract
- Background Arsenic is a toxic and highly abundant metalloid that endangers human health through drinking water and the food chain. The most common forms of arsenic in the environment are arsenate (As(V)) and arsenite (As(III)). As(V) is a non-functional phosphate analog that enters the food chain via plant phosphate transporters. Inside cells, As(V) becomes reduced to As(III) for subsequent extrusion or compartmentation. Although much is known about As(III) transport and handling in microbes and mammals, the transport systems for As(III) have not yet been characterized in plants. Results Here we show that the Nodulin26-like Intrinsic Proteins (NIPs) AtNIP5;1 and AtNIP6;1 from Arabidopsis thaliana, OsNIP2;1 and OsNIP3;2 from Oryza sativa, and LjNIP5;1 and LjNIP6;1 from Lotus japonicus are bi-directional As(III) channels. Expression of these NIPs sensitized yeast cells to As(III) and antimonite (Sb(III)), and direct transport assays confirmed their ability to facilitate As(III) transport across cell membranes. On medium containing As(V), expression of the same NIPs improved yeast growth, probably due to increased As(III) efflux. Our data furthermore provide evidence that NIPs can discriminate between highly similar substrates and that they may have differential preferences in the direction of transport. A subgroup of As(III) permeable channels that group together in a phylogenetic tree required N-terminal truncation for functional expression in yeast. Conclusion This is the first molecular identification of plant As(III) transport systems and we propose that metalloid transport through NIPs is a conserved and ancient feature. Our observations are potentially of great importance for improved remediation and tolerance of plants, and may provide a key to the development of low arsenic crops for food production.
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| 3. |
- Ilina, Yulia, et al.
(author)
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Characterization of the DNA-binding motif of the arsenic-responsive transcription factor Yap8p.
- 2008
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In: The Biochemical journal. - 1470-8728. ; 415:3, s. 467-75
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Journal article (peer-reviewed)abstract
- Saccharomyces cerevisiae uses several mechanisms for arsenic detoxification including the arsenate reductase Acr2p and the arsenite efflux protein Acr3p. ACR2 and ACR3 are transcribed in opposite directions from the same promoter and expression of these genes is regulated by the AP-1 (activator protein 1)-like transcription factor Yap8p. Yap8p has been shown to permanently associate with this promoter and to stimulate ACR2/ACR3 expression in response to arsenic. In the present study we characterized the DNA sequence that is targeted by Yap8p. We show that Yap8p binds to a pseudo-palindromic TGATTAATAATCA sequence that is related to, but distinct from, the sequence recognized by other fungal AP-1 proteins. Probing the promoter by mutational analysis, we confirm the importance of the TTAATAA core element and pin-point nucleotides that flank this element as crucial for Yap8p binding and in vivo activation of ACR3 expression. A genome-wide search for this element combined with global gene expression analysis indicates that the principal function of Yap8p is to control expression of ACR2 and ACR3. We conclude that Yap8p and other yeast AP-1 proteins require distinct DNA-binding motifs to induce gene expression and propose that this fact contributed towards a separation of function between AP-1 proteins during evolution.
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| 6. |
- Kristiansson, Erik, 1978, et al.
(author)
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Evolutionary Forces Act on Promoter Length: Identification of Enriched Cis-Regulatory Elements
- 2009
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In: Molecular Biology and Evolution. ; 26:6, s. 1299-1307
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Journal article (peer-reviewed)abstract
- Transcription factors govern gene expression by binding to short DNA sequences called cis-regulatory elements. These sequences are typically located in promoters, which are regions of variable length upstream of the open reading frames of genes. Here, we report that promoter length and gene function are related in yeast, fungi, and plants. In particular, the promoters for stress-responsive genes are in general longer than those of other genes. Essential genes have, on the other hand, relatively short promoters. We utilize these findings in a novel method for identifying relevant cis-regulatory elements in a set of coexpressed genes. The method is shown to generate more accurate results and fewer false positives compared with other common procedures. Our results suggest that genes with complex transcriptional regulation tend to have longer promoters than genes responding to few signals. This phenomenon is present in all investigated species, indicating that evolution adjust promoter length according to gene function. Identification of cis-regulatory elements in Saccharomyces cerevisiae can be done with the web service located at http://enricher.zool.gu.se.
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| 12. |
- Thorsen, Michael, 1974, et al.
(author)
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Genetic basis of arsenite and cadmium tolerance in Saccharomyces cerevisiae.
- 2009
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In: BMC genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10
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Journal article (peer-reviewed)abstract
- BACKGROUND: Arsenic and cadmium are widely distributed in nature and pose serious threats to the environment and human health. Exposure to these nonessential toxic metals may result in a variety of human diseases including cancer. However, arsenic and cadmium toxicity targets and the cellular systems contributing to tolerance acquisition are not fully known. RESULTS: To gain insight into metal action and cellular tolerance mechanisms, we carried out genome-wide screening of the Saccharomyces cerevisiae haploid and homozygous diploid deletion mutant collections and scored for reduced growth in the presence of arsenite or cadmium. Processes found to be required for tolerance to both metals included sulphur and glutathione biosynthesis, environmental sensing, mRNA synthesis and transcription, and vacuolar/endosomal transport and sorting. We also identified metal-specific defence processes. Arsenite-specific defence functions were related to cell cycle regulation, lipid and fatty acid metabolism, mitochondrial biogenesis, and the cytoskeleton whereas cadmium-specific defence functions were mainly related to sugar/carbohydrate metabolism, and metal-ion homeostasis and transport. Molecular evidence indicated that the cytoskeleton is targeted by arsenite and that phosphorylation of the Snf1p kinase is required for cadmium tolerance. CONCLUSION: This study has pin-pointed core functions that protect cells from arsenite and cadmium toxicity. It also emphasizes the existence of both common and specific defence systems. Since many of the yeast genes that confer tolerance to these agents have homologues in humans, similar biological processes may act in yeast and humans to prevent metal toxicity and carcinogenesis.
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| 13. |
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| 14. |
- Thorsen, Michael, 1974, et al.
(author)
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Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast.
- 2012
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In: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 84:6, s. 1177-88
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Journal article (peer-reviewed)abstract
- Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low-molecular-weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)₃ was also detected and direct transport assays demonstrate that As(GS)₃ does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.
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| 17. |
- Thorsen, Michael, 1974, et al.
(author)
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Quantitative transcriptome, proteome and sulfur metabolite profiling of the Saccharomyces cerevisiae response to arsenite
- 2007
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In: Physiological Genomics. ; 30, s. 35-43
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Journal article (peer-reviewed)abstract
- Arsenic is ubiquitously present in nature, and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative transcriptome, proteome, and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance, and proteolytic activity. Importantly, we observed that nearly all components of the sulfate assimilation and glutathione biosynthesis pathways were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated cellular glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pinpointed transcription factors that mediate the core of the transcriptional response to arsenite. Taken together, our data reveal that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis, and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert.
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| 20. |
- Thorsen, Michael, 1974, et al.
(author)
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The MAPK Hog1p modulates Fps1p-dependent arsenite uptake and tolerance in yeast.
- 2006
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In: Molecular biology of the cell. - 1059-1524 .- 1939-4586. ; 17:10, s. 4400-10
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Journal article (peer-reviewed)abstract
- Arsenic is widely distributed in nature and all organisms possess regulatory mechanisms to evade toxicity and acquire tolerance. Yet, little is known about arsenic sensing and signaling mechanisms or about their impact on tolerance and detoxification systems. Here, we describe a novel role of the S. cerevisiae mitogen-activated protein kinase Hog1p in protecting cells during exposure to arsenite and the related metalloid antimonite. Cells impaired in Hog1p function are metalloid hypersensitive, whereas cells with elevated Hog1p activity display improved tolerance. Hog1p is phosphorylated in response to arsenite and this phosphorylation requires Ssk1p and Pbs2p. Arsenite-activated Hog1p remains primarily cytoplasmic and does not mediate a major transcriptional response. Instead, hog1delta sensitivity is accompanied by elevated cellular arsenic levels and we demonstrate that increased arsenite influx is dependent on the aquaglyceroporin Fps1p. Fps1p is phosphorylated on threonine 231 in vivo and this phosphorylation critically affects Fps1p activity. Moreover, Hog1p is shown to affect Fps1p phosphorylation. Our data are the first to demonstrate Hog1p activation by metalloids and provides a mechanism by which this kinase contributes to tolerance acquisition. Understanding how arsenite/antimonite uptake and toxicity is modulated may prove of value for their use in medical therapy.
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| 21. |
- Wysocki, Robert, et al.
(author)
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Transcriptional Activation of Metalloid Tolerance Genes in Saccharomyces cerevisiae Requires the AP-1like Proteins Yap1p and Yap8p
- 2004
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In: Molecular Biology of the Cell. ; 15:5, s. 2049-2060
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Journal article (peer-reviewed)abstract
- All organisms are equipped with systems for detoxification of the metalloids arsenic and antimony. Here, we show that two parallel pathways involving the AP-1like proteins Yap1p and Yap8p are required for acquisition of metalloid tolerance in the budding yeast S. cerevisiae. Yap8p is demonstrated to reside in the nucleus where it mediates enhanced expression of the arsenic detoxification genes ACR2 and ACR3. Using chromatin immunoprecipitation assays, we show that Yap8p is associated with the ACR3 promoter in untreated as well as arsenic-exposed cells. Like for Yap1p, specific cysteine residues are critical for Yap8p function. We further show that metalloid exposure triggers nuclear accumulation of Yap1p and stimulates expression of antioxidant genes. Yap1p mutants that are unable to accumulate in the nucleus during H2O2 treatment showed nearly normal nuclear retention in response to metalloid exposure. Thus, our data are the first to demonstrate that Yap1p is being regulated by metalloid stress and to indicate that this activation of Yap1p operates in a manner distinct from stress caused by chemical oxidants. We conclude that Yap1p and Yap8p mediate tolerance by controlling separate subsets of detoxification genes and propose that the two AP-1like proteins respond to metalloids through distinct mechanisms.
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