Arsenic and Antimony Transporters in Eukaryotes

Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters

α-Arrestins and Their Functions: From Yeast to Human Health

α-Arrestins, also called arrestin-related trafficking adaptors (ARTs), constitute a large family of proteins conserved from yeast to humans. Despite their evolutionary precedence over their extensively studied relatives of the β-arrestin family, α-arrestins have been discovered relatively recently, and thus their properties are mostly unexplored. The predominant function of α-arrestins is the selective identification of membrane proteins for ubiquitination and degradation, which is an important element in maintaining membrane protein homeostasis as well as global cellular metabolisms. Among members of the arrestin clan, only α-arrestins possess PY motifs that allow canonical binding to WW domains of Rsp5/NEDD4 ubiquitin ligases and the subsequent ubiquitination of membrane proteins leading to their vacuolar/lysosomal degradation. The molecular mechanisms of the selective substrate’s targeting, function, and regulation of α-arrestins in response to different stimuli remain incompletely understood. Several functions of α-arrestins in animal models have been recently characterized, including redox homeostasis regulation, innate immune response regulation, and tumor suppression. However, the molecular mechanisms of α-arrestin regulation and substrate interactions are mainly based on observations from the yeast Saccharomyces cerevisiae model. Nonetheless, α-arrestins have been implicated in health disorders such as diabetes, cardiovascular diseases, neurodegenerative disorders, and tumor progression, placing them in the group of potential therapeutic targets

Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

AbstractThe yeast transporter Acr3p is a low affinity As(III)/H+ and Sb(III)/H+ antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane

The regulatory inputs controlling pleiotropic drug resistance and hypoxic response in yeast converge at the promoter of the aminocholesterol resistance gene RTA1

Aminosterols possessing potent fungicidal activity are attractive alternatives to currently available antifungals. Although their precise mechanism of action is not fully understood, the effect of 7-aminocholesterol (7-ACH) involves a partial block of Δ8-Δ7 isomerase and C-14 reductase. The function of RTA1 encoding the 7-transmembrane helix protein, cloned as the multicopy suppressor of 7-ACH toxicity in yeast, remains unclear. In this report, we show that Rta1p is localized in the plasma membrane and has a high rate of metabolic turnover, as revealed by fluorescence microscopy, cell fractionation and pulse-chase experiments. Analysis of the RTA1-lacZ reporter activity and deletion mapping of the promoter allowed the identification of the regions responsible for negative regulation by Tup1 and the two synergistically acting repressors of hypoxic genes, Rox1p and Mot3p. This was in line with increased RTA1-mediated resistance to 7-ACH under hypoxic conditions, associated with increased Rta1p level. Overexpression of RTA1 also affected the response to the signalling sphingolipid precursor phytosphingosine. Positive inputs of two transcriptional activators Pdr1p and Upc2p were also detected, indicating a regulatory link common to sterol biosynthetic genes as well as those involved in pleiotropic drug resistance and sphingolipid metabolism

Vmr 1p is a novel vacuolar multidrug resistance ABC transporter in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Yhl035p/Vmr1p is an ABC transporter of the MRP subfamily that is conserved in all post Whole Genome Duplication species. The deletion of the YHL035 gene caused growth sensitivity to several amphiphilic drugs such as cycloheximide, 2,4-dichlorophenoxyacetic acid, 2,4-dinitrophenol as well as to cadmium and other toxic metals. Vmr1p-GFP was located in the vacuolar membrane. The ATP-dependent transport of a DNP-S-glutathione conjugate was reduced in a vesicular fraction from the VMR1 deletant. The energy-dependent efflux of rhodamine 6G was increased by VMR1 deletion. Growth sensitivity to cadmium of the VMR1-deleted strain was more pronounced in glycerol/ethanol than in glucose-grown cells. The VMR1 promoter had higher activity when grown in glycerol/ethanol compared with glucose. In glucose, the VMR1 promoter was activated by the deletion of the glucose-dependent repressor ADR1

S129A aSyn forms more inclusions and oligomeric species with higher molecular weight than the WT form.

<p>(A) Intracellular localization of the WT, S129A or S129E aSyn-GFP (left panel) and percentage of yeast cells containing aSyn-GFP inclusions (right panel), assessed by fluorescence microscopy at the indicated time points after aSyn-GFP expression induction (***p<0.001; one way ANOVA with Bonferroni's multiple comparison test). (B) WT, S129A or S129E aSyn-GFP expression and pS129 levels in yeast cells assessed by western blot analysis of total protein extracts, at the indicated time points after aSyn-GFP expression induction (left panel). Densitometric analysis of the immunodetection of aSyn-GFP relative to the intensity obtained for GAPDH, used as loading control, presented in arbitrary units (a.u.) (right panel). Results shown are from one representative experiment from a total of three independent experiments. (C) The aggregated species formed in yeast cells by the WT and S129A aSyn-GFP were resolved using sucrose gradients after 6 hour of aSyn-GFP expression induction. The resulting fractions were separated on an SDS-page gel followed by immunoblotting with an antibody against aSyn (left panel). The same amount of WT and S129A aSyn-GFP protein was applied to the sucrose gradients, as confirmed by western blot (right panel). (D) The aggregated species formed by WT and S129A aSyn-GFP were also resolved using size exclusion-chromatography (SEC) after 6 hours of aSyn-GFP expression induction. Fractions were collected and separated on SDS-PAGE followed by western blot analyses with an antibody against aSyn or pS129 aSyn. The molecular weights indicated correspond to the calibration curve performed in the SEC, as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004302#pgen.1004302.s003" target="_blank">Figure S3</a>. A representative result is shown from at least three independent experiments. Values represent the mean ± SD.</p

The percentage of Triton-X insoluble S129A aSyn increases during clearance.

<p>(A) WT and S129A aSyn-GFP protein levels in yeast cells assessed by western blot analysis of total protein extracts at the indicated time points of aSyn-GFP clearance (left panel). Densitometric analysis of the immunodetection of aSyn relative to the intensity obtained for GAPDH, used as loading control, presented in arbitrary units (a.u.) (right panel) (***p<0.001, *p<0.05; one way ANOVA with Bonferroni's multiple comparison test). (B) Triton-X soluble (TS) and Triton-X insoluble (TI) fractions of aSyn WT or S129A assessed by western blot analysis (upper panel) and determination by densitometric analysis of the percentage of TI aSyn, at the indicated time points of aSyn clearance (lower panel) (*p<0.05; Mann-Whitney test). GAPDH was used as an internal control for the experiment. A representative result is shown from at least four independent experiments. Values represent the mean ± SD.</p