Multilevel Regulation of Membrane Proteins in Response to Metal and Metalloid Stress: A Lesson from Yeast.

In the face of flourishing industrialization and global trade, heavy metal and metalloid contamination of the environment is a growing concern throughout the world. The widespread presence of highly toxic compounds of arsenic, antimony, and cadmium in nature poses a particular threat to human health. Prolonged exposure to these toxins has been associated with severe human diseases, including cancer, diabetes, and neurodegenerative disorders. These toxins are known to induce analogous cellular stresses, such as DNA damage, disturbance of redox homeostasis, and proteotoxicity. To overcome these threats and improve or devise treatment methods, it is crucial to understand the mechanisms of cellular detoxification in metal and metalloid stress. Membrane proteins are key cellular components involved in the uptake, vacuolar/lysosomal sequestration, and efflux of these compounds; thus, deciphering the multilevel regulation of these proteins is of the utmost importance. In this review, we summarize data on the mechanisms of arsenic, antimony, and cadmium detoxification in the context of membrane proteome. We used yeast Saccharomyces cerevisiae as a eukaryotic model to elucidate the complex mechanisms of the production, regulation, and degradation of selected membrane transporters under metal(loid)-induced stress conditions. Additionally, we present data on orthologues membrane proteins involved in metal(loid)-associated diseases in humans.

Identification of amino acid substitutions that toggle substrate selectivity of the yeast arsenite transporter Acr3.

The Acr3 protein family plays a crucial role in metalloid detoxification and includes members from bacteria to higher plants. Most of the Acr3 transporters studied so far are specific for arsenite, whereas Acr3 from budding yeast also shows some capacity to transport antimonite. However, the molecular basis of Acr3 substrate specificity remains poorly understood. By analyzing randomly generated and rationally designed yeast Acr3 variants, critical residues determining substrate specificity were identified for the first time. Replacement of Val173 with Ala abolished antimonite transport without affecting arsenite extrusion. In contrast, substitution of Glu353 with Asp resulted in a loss of arsenite transport activity and a concomitant increase in antimonite translocation capacity. Importantly, Val173 is located close to the hypothetical substrate binding site, whereas Glu353 has been proposed to participate in substrate binding. Identification of key residues conferring substrate selectivity provides a valuable starting point for further studies of the Acr3 family and may have implications for the development of biotechnological applications in metalloid remediation. Moreover, our data contribute to understanding why members of the Acr3 family evolved as arsenite-specific transporters in an environment of ubiquitously present arsenic and trace amounts of antimony.

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 ß-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.

Rsp5-dependent endocytosis and degradation of the arsenite transporter Acr3 requires its N-terminal acidic tail as an endocytic sorting signal and arrestin-related ubiquitin-ligase adaptors.

The yeast plasma membrane transporter Acr3 mediates efflux of toxic arsenite and antimonite. Here, we investigated the mechanisms of Acr3 turnover. We found that after arrival and residence at the plasma membrane, Acr3 is subjected to internalization followed by proteolysis in the vacuole. Endocytic degradation of Acr3 is promoted by the ubiquitin ligase Rsp5 and requires polyubiquitination of Acr3 at multiple lysine residues via lysine 63-linked ubiquitin chains. The turnover of Acr3 also depends on two arrestin-related proteins, Art3/Aly2 and Art4/Rod1, that enable recruitment of Rsp5 to its targets. Finally, we found that a short acidic patch located in the N-terminal tail of Acr3 is needed for its ubiquitination and internalization. We propose that this motif serves as an endocytic signal that facilitates binding of the arrestin-Rsp5 complexes to the Acr3 cargo.

Coupling of RNA polymerase III assembly to cell cycle progression in Saccharomyces cerevisiae.

Assembly of the RNA polymerases in both yeast and humans is proposed to occur in the cytoplasm prior to their nuclear import. Our previous studies identified a cold-sensitive mutation, rpc128-1007, in the yeast gene encoding the second largest Pol III subunit, Rpc128. rpc128-1007 is associated with defective assembly of Pol III complex and, in consequence, decreased level of tRNA synthesis. Here, we show that rpc128-1007 mutant cells remain largely unbudded and larger than wild type cells. Flow cytometry revealed that most rpc128-1007 mutant cells have G1 DNA content, suggesting that this mutation causes pronounced cell cycle delay in the G1 phase. Increased expression of gene encoding Rbs1, the Pol III assembly/import factor, could counteract G1 arrest observed in the rpc128-1007 mutant and restore wild type morphology of mutant cells. Concomitantly, cells lacking Rbs1 show a mild delay in G1 phase exit, indicating that Rbs1 is required for timely cell cycle progression. Using the double rpc128-1007 maf1Δ mutant in which tRNA synthesis is recovered, we confirmed that the Pol III assembly defect associated with rpc128-1007 is a primary cause of cell cycle arrest. Together our results indicate that impairment of Pol III complex assembly is coupled to cell cycle inhibition in the G1 phase.

Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae.

Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H+ and Sb(III)/H+ antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.

Quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride induced alterations in Saccharomyces cerevisiae physiology.

We investigated the influence of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)- N,N,N-trimethyl ammonium chloride) on yeast cells of the parental strain and the IM-resistant mutant (EO25 IMR) growth. The phenotype of this mutant was pleiotropic. The IMR mutant exhibited resistance to ethanol, osmotic shock and oxidative stress, as well as increased sensitivity to UV. Moreover, it was noted that mutant EO25 appears to have an increased resistance to clotrimazole, ketoconazole, fluconazole, nystatin and cycloheximide. It also tolerated growth in the presence of crystal violet, DTT and metals (selenium, tin, arsenic). It was shown that the presence of IM decreased ergosterol level in mutant plasma membrane and increased its unsaturation. These results indicate changes in the cell lipid composition. Western blot analysis showed the induction of Pma1 level by IM. RT-PCR revealed an increased PMA1 expression after IM treatment.

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.

Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼ 90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.

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

The 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.

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.

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.

[Yeast as a model for studying neurodegeneration]. / Drozdze jako model w badaniach chorób neurodegeneracyjnych.

At the level of genetics and physiology the yeast Saccharomyces cerevisiae is are the best characterized eukaryotic cells. The yeast cells can be used as a model to study the mechanisms involved in human disease. Yeast shares conserved cellular mechanisms with all eukaryotes including mammals and human. Nowadays, despite the lack of a neural system, yeasts are successfully used in the study of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease and Parkinson's disease. Exquisite genetics and molecular tools used in biology allow examination of the role of yeast homologues of human genes as well as heterologous expression of human genes in yeast. Yeasts have become a suitable model to study the causes of pathological changes in protein folding, mutations and formation of aggregates.

[Efflux-mediated antimicrobial multidrug resistance]. / Opornosc wielolekowa zwiazana z aktywnym usuwaniem leków z komórek drobnoustrojów.

Multidrug resistance is a major problem in the treatment of infectious diseases caused by bacteria and fungi. One of the basic mechanisms of resistance is active efflux of distinct drugs from cells. Export of toxic compounds from bacterial cells is mediated by proteins of 5 distinct families: MF, SMR, ABC, RND and MATE. The substrate spectrum of efflux pumps includes antibiotics, chemotherapeutics and detergents. Genes that determine resistance can be located on chromosomes or mobile elements (plasmids, transposons, integrons). The presence of resistance genes on mobile elements enables bacteria to transfer those genes between cells and spread the multidrug resistance phenotype. There are several inhibitors of efflux pumps that are currently in the experimental phase. Proteins that mediate multidrug resistance are also present in fungal cells. They belong mainly to the ABC superfamily of transporters and PDR subfamily. These efflux pumps are widely investigated in Saccharomyces cerevisiae.

[The ABC transporters of Saccharomyces cerevisiae]. / ABC transportery Saccharomyces cerevisiae.

The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.

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.

The yeast permease Acr3p is a dual arsenite and antimonite plasma membrane transporter.

The Acr3p permease from the yeast Saccharomyces cerevisiae is a prototype member of the arsenical resistance-3 (Acr3) family of transporters, which are found in all domains of life. Remarkably little is known about substrate specificity, localization and regulation of Acr3 proteins. Here, we show that the yeast Acr3p mediates not only high-level resistance to arsenite but also moderate tolerance to antimonite. The acr3 deletion mutant shows increased sensitivity to antimonite. In addition, overexpression of the ACR3 gene complements antimonite sensitivity of cells lacking the vacuolar ABC transporter Ycf1p. Moreover, both antimonite and arsenite induce transcription of the ACR3 gene resulting in the accumulation of Acr3 transporter at the plasma membrane. However, antimonite is much weaker inducer of the ACR3 gene transcription comparing to arsenite. Interestingly, the presence of metalloids does not influence either stability of Acr3 protein or its intracellular localization suggesting that Acr3p is mainly regulated at the transcriptional level. Finally, transport experiments confirmed that Acr3p indeed mediates efflux of antimonite and thus possesses a dual arsenite and antimonite specificity.

A novel phenotype of eight spores asci in deletants of the prion-like Rnq1p in Saccharomyces cerevisiae.

We report that a null rnq1 mutation in the yeast RNQ1 (YCL028w) prion-like gene of so far unknown function produces the doubling of spores in the asci. This phenotype is possibly due to the lack of inhibition by Rnq1p of an additional mitotic division during ascus formation. This novel phenotype termed "octopus asci" could be similar to prion [PIN+] phenotype.