Direct binding of arsenicals to nuclear transport factors disrupts nucleocytoplasmic transport
Human exposure to the poisonous metalloid arsenic is a global health threat that affects hundreds of millions of people ( ). High concentrations of arsenic in the groundwater have been measured in a large number of countries and long-term exposure is associated with numerous human health problems such as skin disorders, cardiovascular disease, diabetes, cancers of the liver, lung and kidneys, and neurological and neurodegenerative disorders ( ; ; ). At the same time, arsenic-containing compounds are currently used in anticancer and antiparasitic therapy ( ). Pentavalent arsenate, As(V), and trivalent arsenite, As(III) are the most common forms of inorganic arsenic in the environment ( ) and once inside cells, inorganic arsenic can be enzymatically converted into mono-, di-, and trimethylated metabolites ( ). Various forms of arsenic affect cells and living organisms in distinct ways. Due to its chemical similarity to phosphate, As(V) competes with phosphate in biochemical reactions and disrupts adenosine triphosphate (ATP) production. As(III) has high affinity for sulfhydryl groups, such as the thiol groups of cysteine residues, and binding of As(III) and its metabolites to proteins can disrupt protein conformation, function, and interactions ( ; ; ; ; ; ). Methylation affects the toxicity of arsenicals as well as their modes of action and protein binding specificities ( ; ). For example, As(III) can bind up to three cysteine residues, monomethylarsenite [MAs(III)] can bind two cysteine residues and dimethylarsenite [DMAs(III)] can bind only one cysteine residue ( ). Arsenic’s ability to bind to proteins is associated with its toxicity, but also with its therapeutic effects. For instance, binding to cysteine residues in the oncoprotein PML-RARα underlies the anticancer activity of arsenic trioxide in patients with acute promyelocytic leukaemia (APL) ( ; ). Similarly, arsenic-binding to specific kinases and transcriptional regulators is linked to arsenic resistance in yeast and bacteria ( ; ; ). While the toxicity of trivalent arsenite has traditionally been attributed to its interactions with sulfhydryl groups in native (folded) proteins ( ; ), recent studies have shown that As(III) also targets non-native proteins impairing their proper folding ( ; ; ; ). In cells, this results in extensive protein misfolding and aggregation which, in turn, has a negative effect on cell proliferation and viability ( ; ; ; ). Thus, knowledge of arsenic-protein interactions is key to understand the toxic and therapeutic effects of arsenicals as well as cellular sensing and defence mechanisms. To this end, several large-scale studies have been performed with the aim to identify arsenic-binding proteins ( ). For example, 360 proteins bound to arsenic in vitro using a human proteome microarray ( ) and in vivo studies identified 40 arsenic-binding proteins in APL cells ( ), 50 proteins in human breast cancer cells (MCF-7 cell line) ( ), 51 proteins in human embryonic kidney epithelial cells (HEK293T) ( ), and 48 proteins in A549 human lung carcinoma cells ( ). Follow-up experiments indicated that some of these proteins are bona fide targets of arsenic-binding and inhibition ( ; ; ). While identifying the arsenic-binding proteome is a promising approach to address toxicity and resistance mechanisms, the aforementioned in vivo studies identified relatively few targets and a comprehensive catalogue of in vivo arsenic-protein interactions and the resulting consequences on cell physiology is still lacking. In this study, we took a proteomic approach and identified 174 proteins that bind to arsenic in budding yeast Saccharomyces cerevisiae . Proteins involved in nucleocytoplasmic transport were strongly enriched among the arsenic-binding proteins, and data from follow-up experiments is consistent with a model in which arsenic-binding to nuclear transport factors leads to their mislocalization and aggregation, which disrupts protein transport across the nuclear envelope and causes arsenic sensitivity. 

Proteome-wide identification of arsenic-binding proteins in yeast. To identify proteins that bind to arsenic in vivo , we took an unbiased proteomic approach in S. cerevisiae using biotin-conjugated As(III) (hereafter As–biotin) as a probe ( ; ). We previously noted that As-biotin cannot discriminate between As(III)-binding proteins and proteins that bind to MAs(III) due to intracellular conversion of As(III) into MAs(III) ( ; ). Therefore, we incubated yeast cells that lack the methyltransferase enzyme Mtq2 responsible for As(III) methylation ( ) with 50 μM As–biotin without or with a 10 min pretreatment with 1 mM As(III) or 500 μM MAs(III) as blocking agents ( ): the pretreatments were performed to get an indication of which arsenical binds to each protein as binding of As-biotin to a protein is expected to be attenuated in the presence of As(III) or MAs(III), and mtq2Δ cells were used to avoid metabolism of the blocking arsenical ( ; ; ; ). After cell disruption and As-biotin pull-down with streptavidin-agarose beads, candidate arsenic-binding proteins were eluted, separated by SDS-PAGE electrophoresis, and identified by microcapillary liquid chromatograph-tandem mass spectrometry (LC/MS/MS) ( ). As a control, we performed pull-downs using cells that had not been incubated with As-biotin. In total, 776 proteins were identified in at least one of the conditions ( ). To select candidate arsenic-binding proteins, we filtered the 776 proteins using the following criteria: (1) no peptide present in the control, (2) ≥5 unique peptides identified after As-biotin pull-down, and (3) ≥2-fold reduction of signal/peptide intensity when competitor As(III) or MAs(III) was present during pull-down. Applying these stringent filtering criteria gave a list of 174 candidate arsenic-binding proteins ( ). To our knowledge, this represents the largest set of in vivo arsenic-binding proteins reported to date. Several of the 174 yeast proteins have human orthologues that were previously reported to bind to arsenic in large-scale in vitro and in vivo screens ( ; ; ) including subunits of the chaperonin TRiC/CCT complex involved in protein folding, metabolic enzymes such as glycerol-3-phosphate dehydrogenase, aldehyde dehydrogenase and members of the pyruvate dehydrogenase complex, proteins involved in DNA replication including components of the minichromosome maintenance (MCM) complex, α- and β-tubulin, and ribonucleotide reductase implicated in DNA synthesis and repair. Thus, these proteins may represent evolutionarily conserved arsenic-binding targets. Of these, the chaperonin TRiC/CCT ( ), tubulin ( ), and pyruvate dehydrogenase ( ; ) have been proposed to be direct toxicity targets. The majority of the 174 proteins (103 proteins, 59%) had ≥2-fold reduced signal/peptide intensity in the presence of As(III) as well as MAs(III), suggesting that they may bind both arsenicals ( ). 48 proteins (28%) reached the threshold of ≥2-fold reduction in signal/peptide intensity only in presence of MAs(III) while 23 (13%) reached the threshold only in presence of As(III), suggesting that these proteins preferentially bind to either MAs(III) or As(III), respectively. As(III) and MAs(III) preferentially bind to the thiol group of cysteine residues in proteins ( ; ; ), and virtually all 174 proteins (99.4%) contained at least one cysteine compared to 90.7% in the yeast proteome ( P <10 −7 ) ( ). The arsenic-binding set was also significantly enriched for proteins with cysteines adjacent or proximal to other cysteines (CC, CxC, CxxC and CxxxC motifs), and the mean number of cysteines and CC motifs per protein was significantly higher in arsenic-binding proteins compared to the proteome ( ). Additionally, we observed a significant overlap between the arsenic-binding proteins and a set of 145 yeast proteins that possess surface-exposed reactive cysteines (12 proteins, P =0.0002) ( ) ( ). As(III) has been shown to bind to proteins containing zinc finger motifs, specifically to C3H1 and C4 motifs ( ; ). 12 of the 174 proteins ( P =0.11) in our dataset are putative zinc-binding proteins of which 8 are predicted to contain C3H1 and C4 motifs ( ). In sum, the As-biotin probe identified proteins that bind to As(III) and MAs(III) or both arsenicals, and our findings reinforce the strong preference of As(III)/MAs(III) for cysteine residues in proteins in vivo . Protein binding as a possible toxicity mechanism. It has been postulated that trivalent arsenic causes toxicity via protein binding, inactivating or depleting important cellular functions ( ; ; ; ; ; ). However, only few direct toxicity targets and mechanisms have been described to date. One way to pinpoint candidate toxicity targets is to perform drug-induced haploinsufficiency profiling (HIP) assays ( ; ). A previous yeast HIP study identified 33 As(III) sensitive heterozygous diploid knockout mutants ( ) of which 8 encode proteins that were present in the arsenic-binding set ( P <10 −6 ) ( ) including components of TRiC/CCT (Cct1, Cct4, Cct5, Cct7), α-tubulin (Tub3), the nuclear pore protein Nup145, adenine-requiring Ade12, and the serine palmitoyltransferase Lcb1. These proteins may represent bona fide arsenic toxicity targets, as proposed for TRiC/CCT ( ) and tubulin ( ). Another way to identify direct toxicity targets is to integrate chemical-genetic and genetic interaction data ( ). For this, we retrieved negative genetic interactors of selected arsenic-binding proteins and asked whether the sets of negative genetic interactors are enriched for As(III) sensitive mutants. Indeed, we observed significant enrichments in As(III) sensitivity among negative genetic interactors of selected arsenic-binding protein-encoding genes involved in transport across the nuclear envelope ( KAP121/PSE1 ( P =0.0004), NUP84 ( P <10 −20 )), components of TRiC/CCT ( CCT1 ( P <10 −13 ), CCT5 ( P <10 −7 )), the alpha subunit of pyruvate dehydrogenase PDA1 ( P <10 −13 ), the translation regulator GCN20 ( P =0.004), and the serine palmitoyltransferase LCB1 ( P =0.0002) ( ). Thus, the tested arsenic-binding proteins might represent bona fide toxicity targets, as proposed for TRiC/CCT ( ) and pyruvate dehydrogenase ( ; ). We noted that a substantial fraction of the arsenic-binding proteins is essential for cell viability (73 proteins, 42%, P <10 −12 ) ( ), suggesting that arsenic-binding might drain the active pool of these essential proteins, resulting in poor growth or survival of yeast cells during arsenic stress. The set of essential arsenic-binding proteins included proposed toxicity targets such as TRiC/CCT ( ) and tubulin ( ) and novel candidate targets such as proteins involved in nucleocytoplasmic transport ( ; see further). One way arsenic binding affects protein function is by interfering with their folding, thereby preventing proteins from reaching their native fold and hence, their active conformation ( ; ; ; ). We found a significant overlap between the set of arsenic-binding proteins and a set of 143 proteins that aggregated in As(III)-exposed yeast cells (27 proteins, P <10 −16 ) ( ; ) ( ), suggesting that arsenic-binding to these proteins results in their misfolding and aggregation. In sum, our findings suggest that a large fraction of the 174 arsenic-binding proteins identified here may represent bona fide toxicity targets and support the notion that protein binding is a major arsenic toxicity mechanism, by affecting protein folding and/or activity. Thus, integrating arsenic-protein binding data with other datasets is a powerful approach to identify novel toxicity targets. Arsenic binds to proteins involved in nucleocytoplasmic transport. We next addressed if specific categories of protein functions are overrepresented among the arsenic-binding proteins. Gene ontology (GO) analysis revealed that this set was enriched in processes associated with protein import into the nucleus, chaperonin-mediated protein folding, carboxylic acid metabolic processes, nuclear pore localization and organization, DNA unwinding involved in DNA replication, tRNA transport, methylation and aminoacylation, and sphingosine and long-chain fatty acid metabolism ( ). Remarkably, the set of arsenic-binding proteins was markedly enriched for functions in nucleocytoplasmic transport and included 8 out of the 11 importins (Kap104, Kap114, Kap122, Kap123, Kap121/Pse1, Sxm1/Kap108, Nmd5/Kap119, Mtr10/Kap111) present in S. cerevisiae , several exportins (Cse1/Kap109, Crm1/Kap124, Msn5/Kap142), and numerous nuclear pore proteins (Nup84, Nup85, Nup120, Nup133, Nup145, Nup157, Nup170, Nup188, Nup192) ( ). This strong enrichment together with our genetic analyses above implicating some of these proteins as direct toxicity targets (Nup145, Nup84, Kap121/Pse1), raised the possibility that nucleocytoplasmic transport is a key target of arsenic in living cells. Importins are nuclear transport factors that bind to cargo proteins containing nuclear localization signals (NLS) in the cytoplasm and facilitate their passage through the nuclear pore complex (NPC) into the nucleus, exportins mediate the export of cargo proteins back to the cytoplasm, while nuclear pore proteins (Nups) constitute the NPC ( ; ). Importins and exportins belong to the karyopherin (Kap) family of nuclear transport factors. While most karyopherins can bind to their cargos directly (karyopherin β), in the case of the heterodimeric α/β complex, it is karyopherin α that binds to the cargo with karyopherin β stabilizing/enhancing this interaction ( ; ). To validate arsenic-binding to selected importins (karyopherin βs Kap123, Kap121/Pse1, Sxm1/Kap108), exportins (karyopherin βs Crm1/Kap124, Msn5/Kap142) and Nups (Nup84, Nup188), we either introduced plasmids that expressed HA-tagged versions of the corresponding genes into yeast cells or used cells that harboured GFP-tagged versions of the genes in their genomes, and performed As-biotin pulldown assays. All tested proteins bound to As-biotin and this binding was attenuated to various degrees in the presence of competitor As(III) or MAs(III) ( , ). Thus, these proteins directly bind arsenic in vivo in form of As(III) and/or MAs(III). We also chose to test arsenic-binding to the karyopherin α Srp1/Kap60 and the karyopherin β Kap95 even though they were not present in the hit list (no peptides were found for Srp1/Kap60 while Kap95 was below the threshold), because these proteins constitute the heterodimeric α/β complex that plays a key role in nuclear transport of NLS-containing proteins in yeast ( ). Kap95 readily bound to As-biotin and this binding was unaffected by competitor As(III) but clearly attenuated in the presence of MAs(III) ( ). Thus, Kap95 binds to arsenic in form of MAs(III) in vivo . In contrast, Srp1 did not bind to As-biotin ( ). Most of the tested proteins (Sxm1, Kap95, Kap121/Pse1, Msn5, Nup84, Nup188) have adjacent or proximal cysteines in their primary sequence ( ). Analyses of their known or predicted 3D structures revealed that most of these proteins contain cysteine pairs within approximately 5Å of each other ( ), making them suitable substrates for As(III)/MAs(III) in their native folded structures. In contrast, Kap123 and Crm1 lack adjacent or proximal cysteines in their primary sequence, raising the question of how As(III) and/or MAs(III) bind to these proteins. Inspection of their 3D structures revealed the presence of proximal cysteines that could potentially serve as binding sites in both proteins ( ). Finally, Srp1/Kap60 does not have cysteine motifs in its primary sequence and the closest cysteines in its 3D structure are separated by approximately 10Å with the thiol groups pointing in opposite directions ( ), explaining why this protein is a poor substrate for As(III)/MAs(III). Importins mislocalize and aggregate in As(III)-exposed cells. Having established that arsenic binds to individual importins, we next addressed the consequence(s) of this binding. First, we monitored the localization of chromosomally integrated Kap95-GFP. We chose to focus on Kap95 because: (1) it plays a central role in nuclear transport of NLS-containing proteins in yeast ( ), (2) integration of chemical-genetic and genetic interaction data suggested that Kap95 may be a direct arsenic toxicity target ( ), and ( ) the human orthologue of Kap95, Importin 90/KPNB1, was identified as a candidate arsenic-binding protein in MCF-7 cells ( ). In untreated (control) cells, chromosomally integrated Kap95-GFP localized around the yeast nuclear envelope (NE) ( , ). In As(III)-exposed cells, NE localization of Kap95-GFP was disrupted and the protein was instead found in distinct foci that were dispersed throughout the cytosol in the majority of cells ( , ). Cytosolic Kap95-GFP foci were also formed when the translation inhibitor cycloheximide (CHX) was added at the same time as As(III) ( ), suggesting that As(III) impacts the localization of the native (folded) form of Kap95. Kap95 forms a heterodimeric nuclear transport receptor together with Srp1 ( ). As for Kap95, As(III) stress altered the distribution of chromosomally integrated Srp1-GFP from the NE to distinct cytosolic foci ( ) both in the absence and presence of CHX ( ), which is consistent with the two proteins forming a heterodimer. Controlled formation of protein condensates can be used by cells for various physiological purposes whereas aggregation of misfolded proteins represents an irreversible loss of protein function ( ). To address whether formation of cytosolic Kap95-GFP and Srp1-GFP foci are reversible, we monitored their localization after As(III) exposure during 1 h followed by As(III) washout. NE localization of both proteins slowly recovered after As(III) washout, and this recovery largely coincided with the disappearance of cytosolic Kap95-GFP ( ) and Srp1-GFP ( ) foci. CHX, added after the washing step, slowed down or prevented the recovery of Kap95-GFP and Srp1-GFP at the NE as well as the disappearance of cytosolic Kap95-GFP and Srp1-GFP foci ( , ), indicating that foci reversal and signal recovery at the NE require de novo protein synthesis. Thus, formation of Kap95-GFP and Srp1-GFP foci may not be a regulated process that cells use to quickly recover once As(III) stress is relieved. Next, we addressed whether the Kap95-GFP and Srp1-GFP foci represent aggregated forms of these proteins. For this, we isolated total and aggregated proteins by differential centrifugation and separated the proteins in each fraction by SDS-PAGE followed by immunoblotting with an anti-GFP antibody. Both Kap95-GFP and Srp1-GFP were present in the aggregated protein fractions isolated from As(III)-exposed cells while these proteins were largely absent in the aggregated protein fractions of unexposed cells ( ). This finding indicates that Kap95 and Srp1 aggregate in the presence of As(III) and that the cytosolic foci likely represent aggregated forms of these proteins. The data also suggest that arsenic-binding to Kap95, in form of MAs(III), is sufficient to induce mislocalization and aggregation of both Srp1 and Kap95 in the heterodimeric α/β complex. Importantly, the karyopherin βs Kap121/Pse1 ( ) and Kap123 ( ) also aggregated during As(III) exposure. We conclude that arsenic-binding to nuclear import receptors leads to their relocation from the nuclear envelope and subsequent aggregation in the cytosol. As(III) affects Nup localization, NE morphology, and NPC numbers. The arsenic-binding Nups identified in this study are located in the outer ring (Nup84, Nup85, Nup120, Nup133, Nup145) and the inner ring (Nup157, Nup170, Nup188, Nup192) of the NPC ( ). Nup145 is, after proteolytic cleavage, present in the outer ring (Nup145C fragment) and in the NPC core (Nup145N) as one of several so-called FG-Nups that are directly responsible for nucleocytoplasmic transport ( ; ). Similar to the tested importins, As(III) affected the localization of chromosomally integrated Nup84-GFP and Nup188-GFP. In untreated (control) cells, Nup84-GFP was localized in patches in the NE ( ). During As(III) exposure, Nup84-GFP was visible as cytosolic foci and the fluorescence signal appeared to extend from the NE into the cytosol in a substantial fraction of the cells ( , ). Likewise, Nup188-GFP was visible in a punctuate pattern in the NE in unexposed cells whereas cytosolic foci and fluorescence signal extensions from the NE into the cytosol were observed in As(III)-exposed cells ( , ). CHX did not prevent As(III)-induced Nup84-GFP foci formation ( ), suggesting that As(III) affects the localization of the native (folded) form of Nup84. These findings imply that arsenic-binding to Nup84 and Nup188 leads to their mislocalization and that arsenic may cause NE deformations. We next performed immuno- electron microscopy (EM) on untreated and As(III) exposed yeast cells using a primary antibody that detects Nups in conjunction with a secondary 10 nm gold label to simultaneously observe NE morphology, Nup localization, NPC morphology and number, and protein aggregates visible as electron-dense content (EDC) within cells ( ; ). While we did not detect any abnormalities in NPC morphology in As(III)-exposed cells ( ), the exposed cells had fewer visible NPCs per cell section compared to unexposed cells ( ). The lower number of NPCs is probably not a result of reduced Nup levels during As(III) stress as Nup84-GFP and Nup188-GFP levels remained largely unchanged during exposure ( ). Instead, a substantial fraction of Nup immunolabelling was associated with EDCs in As(III)-exposed cells ( ), suggesting that Nups may aggregate. These aggregates were not membrane enclosed, but often localized at sites of NE deformations in which the NE extended into the cytoplasm ( , panel I.) or where the inner and outer leaflets were separated with the outer leaflet extending into the cytoplasm ( , panel II.) forming an outer membrane bud. In sum, arsenic-binding to Nups may result in their mislocalization and aggregation, reducing the number of NPCs present on the NE. Nuclear transport is disrupted during long-term As(III) stress. Having established that importins and Nups mislocalize and aggregate, we next addressed whether nuclear transport is affected in As(III)-exposed cells using established reporters ( ; ). We first monitored the localization of the Srp1/Kap95 substrate GFP-tcNLS (GFP with a tandem classical NLS) under the control of the galactose-inducible GAL1 promoter. Expression of GFP-tcNLS was induced and localization of GFP-tcNLS determined by calculating the ratio of the fluorescence measured in the nucleus over the cytosol (N/C ratio) in the absence or presence of As(III) ( ). Importantly, the N/C ratio significantly decreased in As(III)-exposed cells ( ), suggesting that nuclear accumulation of GFP-tcNLS was inhibited. Similarly, the N/C ratios of two other reporters containing NLS sequences recognized respectively by Kap104 (Nab2NLS-GFP) and Kap121/Pse1 (Pho4NLS-GFP), also decreased in exposed cells. In contrast, the N/C ratio of GFP without an NLS remained largely unaffected during exposure ( ). The total protein levels of Kap95, Srp1 and Kap121/Pse1 were not affected by As(III) ( ). Thus, nuclear import of NLS-containing cargos (GFP-tcNLS, Nab2NLS-GFP, Pho4NLS-GFP) is disrupted in As(III) exposed cells, probably due to arsenic-binding to import receptors and Nups. To address if As(III) also affects nuclear export, we monitored the localization of the transcriptional repressor Mig1-GFP, which is nuclear in the presence of glucose but rapidly exits the nucleus via Msn5-dependent export when glucose is replaced by glycerol ( ). Note that As(III) directly binds to Msn5 ( ). As expected, shifting cells from glucose to glycerol resulted in a substantial drop of nuclear Mig1-GFP within 5 min ( ). When cells were preincubated with As(III) for 1 h, glycerol-stimulated nuclear exit of Mig1-GFP was significantly inhibited ( ). Thus, As(III) also disrupts nuclear export, possibly as a consequence of arsenic binding to export receptors. Previous work has shown that yeast cells depend on transcriptional regulation of genes required for As(III) tolerance ( ). For instance, the transcription factors Yap1 and Msn2 accumulate in the nucleus during As(III) stress where they induce expression of defence genes ( ; ; ) while the transcription factor Sfp1 exits the nucleus upon As(III) exposure which results in down-regulation of protein biosynthesis-related genes ( ). Thus, cells rely on functional nucleocytoplasmic transport to mount an appropriate response to arsenic stress. Nuclear accumulation of chromosomally integrated Yap1-GFP and Msn2-GFP, as well as nuclear exit of Sfp1-GFP was efficient within 5–15 min of As(III) exposure ( ), consistent with their role as As(III)-responsive factors. These data suggest that nuclear protein import and export is not affected during short-term exposure, possibly because the arsenic that enters cells is first recognized by arsenic-specific sensing and signalling systems before sufficient arsenic accumulates to poison cellular functions. Alternatively, these stress-responsive transcription factors might cross the NE through As(III)-insensitive pathways. Collectively, our data suggest that nuclear transport is disrupted during long-term As(III) stress but remains functional in the initial phase of exposure. Our chemical-genetic and genetic interaction data ( ) suggested that arsenic-binding to proteins mediating nucleocytoplasmic transport causes toxicity. In further support of this notion, cells with weakened (temperature-sensitive) alleles of Kap95 ( kap95 -L63A) and Kap121/Pse1 ( kap121 -Δ34) ( ) were As(III) sensitive ( ). Thus, disruption of nucleocytoplasmic transport may result in arsenic sensitivity. 

To identify proteins that bind to arsenic in vivo , we took an unbiased proteomic approach in S. cerevisiae using biotin-conjugated As(III) (hereafter As–biotin) as a probe ( ; ). We previously noted that As-biotin cannot discriminate between As(III)-binding proteins and proteins that bind to MAs(III) due to intracellular conversion of As(III) into MAs(III) ( ; ). Therefore, we incubated yeast cells that lack the methyltransferase enzyme Mtq2 responsible for As(III) methylation ( ) with 50 μM As–biotin without or with a 10 min pretreatment with 1 mM As(III) or 500 μM MAs(III) as blocking agents ( ): the pretreatments were performed to get an indication of which arsenical binds to each protein as binding of As-biotin to a protein is expected to be attenuated in the presence of As(III) or MAs(III), and mtq2Δ cells were used to avoid metabolism of the blocking arsenical ( ; ; ; ). After cell disruption and As-biotin pull-down with streptavidin-agarose beads, candidate arsenic-binding proteins were eluted, separated by SDS-PAGE electrophoresis, and identified by microcapillary liquid chromatograph-tandem mass spectrometry (LC/MS/MS) ( ). As a control, we performed pull-downs using cells that had not been incubated with As-biotin. In total, 776 proteins were identified in at least one of the conditions ( ). To select candidate arsenic-binding proteins, we filtered the 776 proteins using the following criteria: (1) no peptide present in the control, (2) ≥5 unique peptides identified after As-biotin pull-down, and (3) ≥2-fold reduction of signal/peptide intensity when competitor As(III) or MAs(III) was present during pull-down. Applying these stringent filtering criteria gave a list of 174 candidate arsenic-binding proteins ( ). To our knowledge, this represents the largest set of in vivo arsenic-binding proteins reported to date. Several of the 174 yeast proteins have human orthologues that were previously reported to bind to arsenic in large-scale in vitro and in vivo screens ( ; ; ) including subunits of the chaperonin TRiC/CCT complex involved in protein folding, metabolic enzymes such as glycerol-3-phosphate dehydrogenase, aldehyde dehydrogenase and members of the pyruvate dehydrogenase complex, proteins involved in DNA replication including components of the minichromosome maintenance (MCM) complex, α- and β-tubulin, and ribonucleotide reductase implicated in DNA synthesis and repair. Thus, these proteins may represent evolutionarily conserved arsenic-binding targets. Of these, the chaperonin TRiC/CCT ( ), tubulin ( ), and pyruvate dehydrogenase ( ; ) have been proposed to be direct toxicity targets. The majority of the 174 proteins (103 proteins, 59%) had ≥2-fold reduced signal/peptide intensity in the presence of As(III) as well as MAs(III), suggesting that they may bind both arsenicals ( ). 48 proteins (28%) reached the threshold of ≥2-fold reduction in signal/peptide intensity only in presence of MAs(III) while 23 (13%) reached the threshold only in presence of As(III), suggesting that these proteins preferentially bind to either MAs(III) or As(III), respectively. As(III) and MAs(III) preferentially bind to the thiol group of cysteine residues in proteins ( ; ; ), and virtually all 174 proteins (99.4%) contained at least one cysteine compared to 90.7% in the yeast proteome ( P <10 −7 ) ( ). The arsenic-binding set was also significantly enriched for proteins with cysteines adjacent or proximal to other cysteines (CC, CxC, CxxC and CxxxC motifs), and the mean number of cysteines and CC motifs per protein was significantly higher in arsenic-binding proteins compared to the proteome ( ). Additionally, we observed a significant overlap between the arsenic-binding proteins and a set of 145 yeast proteins that possess surface-exposed reactive cysteines (12 proteins, P =0.0002) ( ) ( ). As(III) has been shown to bind to proteins containing zinc finger motifs, specifically to C3H1 and C4 motifs ( ; ). 12 of the 174 proteins ( P =0.11) in our dataset are putative zinc-binding proteins of which 8 are predicted to contain C3H1 and C4 motifs ( ). In sum, the As-biotin probe identified proteins that bind to As(III) and MAs(III) or both arsenicals, and our findings reinforce the strong preference of As(III)/MAs(III) for cysteine residues in proteins in vivo . 

It has been postulated that trivalent arsenic causes toxicity via protein binding, inactivating or depleting important cellular functions ( ; ; ; ; ; ). However, only few direct toxicity targets and mechanisms have been described to date. One way to pinpoint candidate toxicity targets is to perform drug-induced haploinsufficiency profiling (HIP) assays ( ; ). A previous yeast HIP study identified 33 As(III) sensitive heterozygous diploid knockout mutants ( ) of which 8 encode proteins that were present in the arsenic-binding set ( P <10 −6 ) ( ) including components of TRiC/CCT (Cct1, Cct4, Cct5, Cct7), α-tubulin (Tub3), the nuclear pore protein Nup145, adenine-requiring Ade12, and the serine palmitoyltransferase Lcb1. These proteins may represent bona fide arsenic toxicity targets, as proposed for TRiC/CCT ( ) and tubulin ( ). Another way to identify direct toxicity targets is to integrate chemical-genetic and genetic interaction data ( ). For this, we retrieved negative genetic interactors of selected arsenic-binding proteins and asked whether the sets of negative genetic interactors are enriched for As(III) sensitive mutants. Indeed, we observed significant enrichments in As(III) sensitivity among negative genetic interactors of selected arsenic-binding protein-encoding genes involved in transport across the nuclear envelope ( KAP121/PSE1 ( P =0.0004), NUP84 ( P <10 −20 )), components of TRiC/CCT ( CCT1 ( P <10 −13 ), CCT5 ( P <10 −7 )), the alpha subunit of pyruvate dehydrogenase PDA1 ( P <10 −13 ), the translation regulator GCN20 ( P =0.004), and the serine palmitoyltransferase LCB1 ( P =0.0002) ( ). Thus, the tested arsenic-binding proteins might represent bona fide toxicity targets, as proposed for TRiC/CCT ( ) and pyruvate dehydrogenase ( ; ). We noted that a substantial fraction of the arsenic-binding proteins is essential for cell viability (73 proteins, 42%, P <10 −12 ) ( ), suggesting that arsenic-binding might drain the active pool of these essential proteins, resulting in poor growth or survival of yeast cells during arsenic stress. The set of essential arsenic-binding proteins included proposed toxicity targets such as TRiC/CCT ( ) and tubulin ( ) and novel candidate targets such as proteins involved in nucleocytoplasmic transport ( ; see further). One way arsenic binding affects protein function is by interfering with their folding, thereby preventing proteins from reaching their native fold and hence, their active conformation ( ; ; ; ). We found a significant overlap between the set of arsenic-binding proteins and a set of 143 proteins that aggregated in As(III)-exposed yeast cells (27 proteins, P <10 −16 ) ( ; ) ( ), suggesting that arsenic-binding to these proteins results in their misfolding and aggregation. In sum, our findings suggest that a large fraction of the 174 arsenic-binding proteins identified here may represent bona fide toxicity targets and support the notion that protein binding is a major arsenic toxicity mechanism, by affecting protein folding and/or activity. Thus, integrating arsenic-protein binding data with other datasets is a powerful approach to identify novel toxicity targets. 

We next addressed if specific categories of protein functions are overrepresented among the arsenic-binding proteins. Gene ontology (GO) analysis revealed that this set was enriched in processes associated with protein import into the nucleus, chaperonin-mediated protein folding, carboxylic acid metabolic processes, nuclear pore localization and organization, DNA unwinding involved in DNA replication, tRNA transport, methylation and aminoacylation, and sphingosine and long-chain fatty acid metabolism ( ). Remarkably, the set of arsenic-binding proteins was markedly enriched for functions in nucleocytoplasmic transport and included 8 out of the 11 importins (Kap104, Kap114, Kap122, Kap123, Kap121/Pse1, Sxm1/Kap108, Nmd5/Kap119, Mtr10/Kap111) present in S. cerevisiae , several exportins (Cse1/Kap109, Crm1/Kap124, Msn5/Kap142), and numerous nuclear pore proteins (Nup84, Nup85, Nup120, Nup133, Nup145, Nup157, Nup170, Nup188, Nup192) ( ). This strong enrichment together with our genetic analyses above implicating some of these proteins as direct toxicity targets (Nup145, Nup84, Kap121/Pse1), raised the possibility that nucleocytoplasmic transport is a key target of arsenic in living cells. Importins are nuclear transport factors that bind to cargo proteins containing nuclear localization signals (NLS) in the cytoplasm and facilitate their passage through the nuclear pore complex (NPC) into the nucleus, exportins mediate the export of cargo proteins back to the cytoplasm, while nuclear pore proteins (Nups) constitute the NPC ( ; ). Importins and exportins belong to the karyopherin (Kap) family of nuclear transport factors. While most karyopherins can bind to their cargos directly (karyopherin β), in the case of the heterodimeric α/β complex, it is karyopherin α that binds to the cargo with karyopherin β stabilizing/enhancing this interaction ( ; ). To validate arsenic-binding to selected importins (karyopherin βs Kap123, Kap121/Pse1, Sxm1/Kap108), exportins (karyopherin βs Crm1/Kap124, Msn5/Kap142) and Nups (Nup84, Nup188), we either introduced plasmids that expressed HA-tagged versions of the corresponding genes into yeast cells or used cells that harboured GFP-tagged versions of the genes in their genomes, and performed As-biotin pulldown assays. All tested proteins bound to As-biotin and this binding was attenuated to various degrees in the presence of competitor As(III) or MAs(III) ( , ). Thus, these proteins directly bind arsenic in vivo in form of As(III) and/or MAs(III). We also chose to test arsenic-binding to the karyopherin α Srp1/Kap60 and the karyopherin β Kap95 even though they were not present in the hit list (no peptides were found for Srp1/Kap60 while Kap95 was below the threshold), because these proteins constitute the heterodimeric α/β complex that plays a key role in nuclear transport of NLS-containing proteins in yeast ( ). Kap95 readily bound to As-biotin and this binding was unaffected by competitor As(III) but clearly attenuated in the presence of MAs(III) ( ). Thus, Kap95 binds to arsenic in form of MAs(III) in vivo . In contrast, Srp1 did not bind to As-biotin ( ). Most of the tested proteins (Sxm1, Kap95, Kap121/Pse1, Msn5, Nup84, Nup188) have adjacent or proximal cysteines in their primary sequence ( ). Analyses of their known or predicted 3D structures revealed that most of these proteins contain cysteine pairs within approximately 5Å of each other ( ), making them suitable substrates for As(III)/MAs(III) in their native folded structures. In contrast, Kap123 and Crm1 lack adjacent or proximal cysteines in their primary sequence, raising the question of how As(III) and/or MAs(III) bind to these proteins. Inspection of their 3D structures revealed the presence of proximal cysteines that could potentially serve as binding sites in both proteins ( ). Finally, Srp1/Kap60 does not have cysteine motifs in its primary sequence and the closest cysteines in its 3D structure are separated by approximately 10Å with the thiol groups pointing in opposite directions ( ), explaining why this protein is a poor substrate for As(III)/MAs(III). 

Having established that arsenic binds to individual importins, we next addressed the consequence(s) of this binding. First, we monitored the localization of chromosomally integrated Kap95-GFP. We chose to focus on Kap95 because: (1) it plays a central role in nuclear transport of NLS-containing proteins in yeast ( ), (2) integration of chemical-genetic and genetic interaction data suggested that Kap95 may be a direct arsenic toxicity target ( ), and ( ) the human orthologue of Kap95, Importin 90/KPNB1, was identified as a candidate arsenic-binding protein in MCF-7 cells ( ). In untreated (control) cells, chromosomally integrated Kap95-GFP localized around the yeast nuclear envelope (NE) ( , ). In As(III)-exposed cells, NE localization of Kap95-GFP was disrupted and the protein was instead found in distinct foci that were dispersed throughout the cytosol in the majority of cells ( , ). Cytosolic Kap95-GFP foci were also formed when the translation inhibitor cycloheximide (CHX) was added at the same time as As(III) ( ), suggesting that As(III) impacts the localization of the native (folded) form of Kap95. Kap95 forms a heterodimeric nuclear transport receptor together with Srp1 ( ). As for Kap95, As(III) stress altered the distribution of chromosomally integrated Srp1-GFP from the NE to distinct cytosolic foci ( ) both in the absence and presence of CHX ( ), which is consistent with the two proteins forming a heterodimer. Controlled formation of protein condensates can be used by cells for various physiological purposes whereas aggregation of misfolded proteins represents an irreversible loss of protein function ( ). To address whether formation of cytosolic Kap95-GFP and Srp1-GFP foci are reversible, we monitored their localization after As(III) exposure during 1 h followed by As(III) washout. NE localization of both proteins slowly recovered after As(III) washout, and this recovery largely coincided with the disappearance of cytosolic Kap95-GFP ( ) and Srp1-GFP ( ) foci. CHX, added after the washing step, slowed down or prevented the recovery of Kap95-GFP and Srp1-GFP at the NE as well as the disappearance of cytosolic Kap95-GFP and Srp1-GFP foci ( , ), indicating that foci reversal and signal recovery at the NE require de novo protein synthesis. Thus, formation of Kap95-GFP and Srp1-GFP foci may not be a regulated process that cells use to quickly recover once As(III) stress is relieved. Next, we addressed whether the Kap95-GFP and Srp1-GFP foci represent aggregated forms of these proteins. For this, we isolated total and aggregated proteins by differential centrifugation and separated the proteins in each fraction by SDS-PAGE followed by immunoblotting with an anti-GFP antibody. Both Kap95-GFP and Srp1-GFP were present in the aggregated protein fractions isolated from As(III)-exposed cells while these proteins were largely absent in the aggregated protein fractions of unexposed cells ( ). This finding indicates that Kap95 and Srp1 aggregate in the presence of As(III) and that the cytosolic foci likely represent aggregated forms of these proteins. The data also suggest that arsenic-binding to Kap95, in form of MAs(III), is sufficient to induce mislocalization and aggregation of both Srp1 and Kap95 in the heterodimeric α/β complex. Importantly, the karyopherin βs Kap121/Pse1 ( ) and Kap123 ( ) also aggregated during As(III) exposure. We conclude that arsenic-binding to nuclear import receptors leads to their relocation from the nuclear envelope and subsequent aggregation in the cytosol. 

The arsenic-binding Nups identified in this study are located in the outer ring (Nup84, Nup85, Nup120, Nup133, Nup145) and the inner ring (Nup157, Nup170, Nup188, Nup192) of the NPC ( ). Nup145 is, after proteolytic cleavage, present in the outer ring (Nup145C fragment) and in the NPC core (Nup145N) as one of several so-called FG-Nups that are directly responsible for nucleocytoplasmic transport ( ; ). Similar to the tested importins, As(III) affected the localization of chromosomally integrated Nup84-GFP and Nup188-GFP. In untreated (control) cells, Nup84-GFP was localized in patches in the NE ( ). During As(III) exposure, Nup84-GFP was visible as cytosolic foci and the fluorescence signal appeared to extend from the NE into the cytosol in a substantial fraction of the cells ( , ). Likewise, Nup188-GFP was visible in a punctuate pattern in the NE in unexposed cells whereas cytosolic foci and fluorescence signal extensions from the NE into the cytosol were observed in As(III)-exposed cells ( , ). CHX did not prevent As(III)-induced Nup84-GFP foci formation ( ), suggesting that As(III) affects the localization of the native (folded) form of Nup84. These findings imply that arsenic-binding to Nup84 and Nup188 leads to their mislocalization and that arsenic may cause NE deformations. We next performed immuno- electron microscopy (EM) on untreated and As(III) exposed yeast cells using a primary antibody that detects Nups in conjunction with a secondary 10 nm gold label to simultaneously observe NE morphology, Nup localization, NPC morphology and number, and protein aggregates visible as electron-dense content (EDC) within cells ( ; ). While we did not detect any abnormalities in NPC morphology in As(III)-exposed cells ( ), the exposed cells had fewer visible NPCs per cell section compared to unexposed cells ( ). The lower number of NPCs is probably not a result of reduced Nup levels during As(III) stress as Nup84-GFP and Nup188-GFP levels remained largely unchanged during exposure ( ). Instead, a substantial fraction of Nup immunolabelling was associated with EDCs in As(III)-exposed cells ( ), suggesting that Nups may aggregate. These aggregates were not membrane enclosed, but often localized at sites of NE deformations in which the NE extended into the cytoplasm ( , panel I.) or where the inner and outer leaflets were separated with the outer leaflet extending into the cytoplasm ( , panel II.) forming an outer membrane bud. In sum, arsenic-binding to Nups may result in their mislocalization and aggregation, reducing the number of NPCs present on the NE. 

Having established that importins and Nups mislocalize and aggregate, we next addressed whether nuclear transport is affected in As(III)-exposed cells using established reporters ( ; ). We first monitored the localization of the Srp1/Kap95 substrate GFP-tcNLS (GFP with a tandem classical NLS) under the control of the galactose-inducible GAL1 promoter. Expression of GFP-tcNLS was induced and localization of GFP-tcNLS determined by calculating the ratio of the fluorescence measured in the nucleus over the cytosol (N/C ratio) in the absence or presence of As(III) ( ). Importantly, the N/C ratio significantly decreased in As(III)-exposed cells ( ), suggesting that nuclear accumulation of GFP-tcNLS was inhibited. Similarly, the N/C ratios of two other reporters containing NLS sequences recognized respectively by Kap104 (Nab2NLS-GFP) and Kap121/Pse1 (Pho4NLS-GFP), also decreased in exposed cells. In contrast, the N/C ratio of GFP without an NLS remained largely unaffected during exposure ( ). The total protein levels of Kap95, Srp1 and Kap121/Pse1 were not affected by As(III) ( ). Thus, nuclear import of NLS-containing cargos (GFP-tcNLS, Nab2NLS-GFP, Pho4NLS-GFP) is disrupted in As(III) exposed cells, probably due to arsenic-binding to import receptors and Nups. To address if As(III) also affects nuclear export, we monitored the localization of the transcriptional repressor Mig1-GFP, which is nuclear in the presence of glucose but rapidly exits the nucleus via Msn5-dependent export when glucose is replaced by glycerol ( ). Note that As(III) directly binds to Msn5 ( ). As expected, shifting cells from glucose to glycerol resulted in a substantial drop of nuclear Mig1-GFP within 5 min ( ). When cells were preincubated with As(III) for 1 h, glycerol-stimulated nuclear exit of Mig1-GFP was significantly inhibited ( ). Thus, As(III) also disrupts nuclear export, possibly as a consequence of arsenic binding to export receptors. Previous work has shown that yeast cells depend on transcriptional regulation of genes required for As(III) tolerance ( ). For instance, the transcription factors Yap1 and Msn2 accumulate in the nucleus during As(III) stress where they induce expression of defence genes ( ; ; ) while the transcription factor Sfp1 exits the nucleus upon As(III) exposure which results in down-regulation of protein biosynthesis-related genes ( ). Thus, cells rely on functional nucleocytoplasmic transport to mount an appropriate response to arsenic stress. Nuclear accumulation of chromosomally integrated Yap1-GFP and Msn2-GFP, as well as nuclear exit of Sfp1-GFP was efficient within 5–15 min of As(III) exposure ( ), consistent with their role as As(III)-responsive factors. These data suggest that nuclear protein import and export is not affected during short-term exposure, possibly because the arsenic that enters cells is first recognized by arsenic-specific sensing and signalling systems before sufficient arsenic accumulates to poison cellular functions. Alternatively, these stress-responsive transcription factors might cross the NE through As(III)-insensitive pathways. Collectively, our data suggest that nuclear transport is disrupted during long-term As(III) stress but remains functional in the initial phase of exposure. Our chemical-genetic and genetic interaction data ( ) suggested that arsenic-binding to proteins mediating nucleocytoplasmic transport causes toxicity. In further support of this notion, cells with weakened (temperature-sensitive) alleles of Kap95 ( kap95 -L63A) and Kap121/Pse1 ( kap121 -Δ34) ( ) were As(III) sensitive ( ). Thus, disruption of nucleocytoplasmic transport may result in arsenic sensitivity. 

This current study strongly implicates nucleocytoplasmic transport as a key target of arsenic toxicity. First, our proteome-wide approach identified 174 arsenic-binding proteins in vivo , of which proteins involved in nucleocytoplasmic transport were remarkably enriched. In fact, arsenic bound to most of the importins present in S. cerevisiae and we verified arsenic-binding to selected importins, exportins, and Nups. Second, we demonstrated that importins and Nups mislocalized and aggregated, and that the number of NPCs was reduced in As(III) exposed cells. Third, we provided evidence that nucleocytoplasmic transport is impaired during As(III) exposure and that cells with defective nuclear protein transport function are As(III) sensitive. Together, our data are consistent with a model in which arsenic-binding to nuclear transport factors leads to their mislocalization and aggregation, disrupting nucleocytoplasmic transport and causing As(III) sensitivity. Previous in vivo proteome-wide studies using the As-biotin probe in various human cell lines typically identified 40–50 candidate arsenic-binding proteins ( ; ; ; ). Our current study yielded 174 proteins representing, to our knowledge, the largest set of in vivo arsenic-binding targets reported to date. Some proteins known to bind to arsenic were absent from our hit list, including the As(III)-sensing transcription factor Yap8 ( ). Low-abundance proteins are less represented in our dataset, and some true targets might be missed due to the stringent filtering criteria used. A study that applied As-biotin to a human proteome microarray identified 360 candidate arsenic-binding proteins ( ). It is unclear whether proteins are properly folded on the microarray, which is an important aspect since non-native structures could result in cysteine residues being more easily accessible for arsenic-binding than in folded native proteins. Nevertheless, the authors demonstrated that one of the 360 candidate proteins, hexokinase 2, is a direct target of arsenic inhibition in vivo ( ). Collectively, these studies demonstrate the great utility of the As-biotin probe for proteome-wide identification of binding and toxicity targets, and together provide a valuable resource for mechanistic studies of on the toxicity, pathology, and therapeutic effects of arsenicals. While As-biotin is a valuable tool, it cannot discriminate between As(III)- and MAs(III)-binding proteins ( ). By using mtq2Δ cells and pretreatments with As(III) and MAs(III) as blocking agents, we identified proteins that bound to either As(III) or MAs(III), or to both arsenicals ( ). For example, Kap121/Pse1 and Crm1 bound to both arsenicals whilst Kap95 primarily bound to MAs(III) ( ). Interestingly, previous studies indicated that As(III) and MAs(III) can bind to distinct sets of cysteine thiols in target proteins thereby eliciting stress-specific responses ( ). How specific cysteine residues distinguish between As(III) and MAs(III) is not known. While our study suggests that the majority of the 174 arsenic-binding proteins can bind to As(III) as well as MAs(III), the exact form of arsenic, the residues involved, and the physiological consequences of the binding remain to be defined. The observations that As(III)-induced Kap95-GFP and Nup84-GFP foci formation were unaffected in the presence of CHX ( , ), suggests that arsenic targets the native (folded) form of these proteins promoting their mislocalization and aggregation ( , , ). Previously, we showed that As(III) treatment of yeast cells resulted in the formation of Hsp104-GFP foci that could be prevented by CHX ( ; ; ; ). Based on these and other findings, we concluded that As(III) primarily targets non-native proteins for misfolding and aggregation ( ; ; ; ). It is important to note that there is a fundamental difference between Hsp104-GFP foci and Kap95-GFP foci that form in As(III)-exposed cells. Hsp104 is a disaggregase that associates with and reactivates aggregated proteins in S. cerevisiae ( ) and Hsp104-GFP is a well-established marker for cytosolic protein aggregation ( ; ; ). Thus, As(III)-induced Hsp104-GFP foci formation is a consequence of global aggregation of cytosolic proteins to which Hsp104 associates while its own activity is unaffected ( ; ). In contrast, Kap95-GFP foci represents aggregated ( ) and probably unfunctional Kap95 since nuclear import of the Kap95/Srp1 cargo GFP-tcNLS was impaired in As(III)-treated cells ( ). Moreover, unlike Hsp104 and it cochaperones, karyopherins are unlikely to recognize misfolded proteins. Thus, native and functional Hsp104-GFP forms foci by associating with non-native proteins while Kap95-GFP (and probably also Nup84-GFP) most likely forms foci because it aggregates. Hence, arsenic can directly modify cysteines in non-native ( ; ) as well as native (this work) proteins driving their unfolding and aggregation, with non-native proteins being particularly vulnerable ( ; ). This raises the question if some (or many) of the proteins isolated in the As-biotin experiments are being brought down indirectly in aggregates. However, the As-biotin pull-down experiments would probably not include aggregates because the beads are centrifuged at 1000 × g , which is not nearly high enough to sediment aggregates. This is also consistent with the relatively low level of overlap between the proteins identified as arsenic-binding and those identified by sedimentation of As(III)-induced aggregates ( ). It has been shown that cytosolic protein aggregates can impair nucleocytoplasmic transport by sequestering nuclear shuttle factors ( ). While As(III) induces global aggregation of cytosolic proteins in yeast ( ), our data point to a more direct mechanism where arsenic impairs nucleocytoplasmic transport by binding to nuclear import and export factors. Arsenic may also affect nucleocytoplasmic transport by binding to Nups, which then leads to reduced number of NPCs in the NE ( ). Additionally, As(III) induces morphological abnormalities of the NE with deformations extending into the cytosol ( ). Interestingly, genetic perturbations that result in NE aberrations, such as NE protrusions that extend into the cytosol, are linked to loss-of-function mutations of NPC components ( ) including several Nups identified in our study ( e.g . Nup85, Nup188, Nup145). Thus, the observed NE aberration during As(III) stress may be a direct consequence of arsenic-binding to Nups, interfering with their function. Several studies have linked arsenic exposure to an increased prevalence of neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease ( ; ). These diseases are characterized by the pathological accumulation of protein aggregates ( ), and there is growing evidence that arsenic contributes to these diseases by impairing protein folding in cells ( ). Interestingly, mislocalization of nucleocytoplasmic transport factors, such as Nups and importins, and the disruption of nucleocytoplasmic transport have been implicated in the pathology of neurodegenerative disorders ( ; ). Thus, the observed impairment of nucleocytoplasmic transport by arsenic, via direct binding, mislocalization, and aggregation of individual nuclear transport factors described in this current study, may be an additional mechanism by which this metalloid contributes to pathology. In conclusion, our study provides novel insights into the molecular mechanisms by which arsenic disrupts cellular function, specifically its impact on nucleocytoplasmic transport. These findings have broad implications for understanding how environmental poisons affect cells and organisms and may serve as a basis for future research on the toxic and therapeutic effects of arsenicals. 

Yeast strains, plasmids, and culturing conditions. All yeast strains and plasmids used in this work are listed in . The S. cerevisiae strains are based on BY4741 ( ), the deletion collection ( ), the collection of temperature-sensitive mutants of essential yeast genes ( ), and the GFP collection ( ). The strains harbouring nuclear transport reporters have been described previously ( ). Plasmids containing HA-tagged versions of Kap123, Sxm1, Nup84, and Nup188 were constructed via Gateway Recombination Cloning (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Gene sequences were amplified by PCR using genomic DNA as template, inserted into the donor plasmid pDONR221 (Thermo Fisher Scientific, Waltham, MA, USA) and then into the destination vector pAG426GPD-ccdB-HA (Addgene plasmid #14252; http://n2t.net/addgene:14252 ) ( ). All plasmids were verified by sequencing. Cells were routinely grown at 30°C in minimal YNB (yeast nitrogen base) medium with 2% glucose as carbon source. Cells containing nuclear transport reporters were grown with 2% raffinose as carbon source and protein expression was induced with 0.5% galactose for 4 h. For growth assays on plates, cells were grown until log phase, the optical density (OD) at 600 nm adjusted to equal for all cultures, diluted in 10-fold steps, plated using a sterilized stamp, and incubated for 2–3 days at 30°C. Where indicated, the following chemicals were added: sodium arsenite (NaAsO 2 , S7400), cycloheximide (C7698) (both from Sigma-Aldrich, St. Louis, MO, USA), biotinyl p-aminophenyl arsenic acid (As-biotin, B394970; Toronto Research Chemicals, North York, Canada), and monomethylarsonous acid (CH 5 AsO 2 , M565100; LGC Standards, Wesel, Germany). Arsenic-binding assays. Proteome-wide identification of arsenic-binding proteins was performed with mtq2Δ cells grown to early log phase in YNB medium. For the last hour, the medium was switched to YEPD (Yeast Extract Peptone Dextrose) with 2% glucose and split into control and treatment cultures. The cultures were pretreated for 10 min with either As(III) or MAs(III) as blocking agents, followed by 10 min incubation with As-biotin at 30°C. The cultures were collected, pelleted, and frozen at −80°C or lysed directly. Cell lysis was performed by bead beating (1 min, 4°C) in IP buffer (1x TNT buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton, pH 7–7.5)), 1x protease inhibitor (Complete mini, EDTA-free; Roche), 1x phosphatase inhibitor (PhosSTOP Easypack; Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Streptavidin agarose beads (ThermoFisher, 20353) were prepared by a 1x TNT buffer wash and aliquoted to all lysates. After a 1 h incubation (4°C, rotating), pull-down was performed by centrifugation (1000× g , 1 min) and washing three times in 1x TNT. Proteins present in the pull-down were separated by SDS-PAGE electrophoresis followed by identification using microcapillary liquid chromatograph-tandem mass spectrometry (LC/MS/MS) at the Taplin Mass Spectrometry Facility at Harvard Medical School. To validate arsenic-binding to selected proteins, we used wild type cells carrying plasmids with HA-tagged versions of the corresponding genes or cells that harboured GFP-tagged versions of the genes in their genomes. After As-biotin pull-down and SDS-PAGE as described above, the proteins were visualized by Western blotting using anti-GFP rabbit IgG (1:8000, A11122; Invitrogen, Waltham, MA, USA) or anti-HA mouse IgG (1:1000, sc-7392; Santa Cruz Biotechnologies, Dallas, TX, USA) primary antibodies, and anti-rabbit IgG (1:3000, 84564) and anti-mouse IgG (1 : 5000, 84545) secondary antibodies (both from Invitrogen (Waltham, MA, USA)). A more extensive description of the Western blot protocol is provided in the section ‘ ’ below. Bioinformatics and protein structure analyses. Negative genetic interactors (including negative genetic, synthetic growth defect, synthetic lethality) of selected arsenic-binding hits were retrieved from the Saccharomyces genome database (SGD) ( ) and compared to a compendium of 712 As(III) sensitive S. cerevisiae mutants that contains the genes identified at least once in four previous genome-wide phenotypic screens ( ; ; ; ). The significance of the overlaps between datasets was calculated by the hyper-geometric test. All datasets used for comparisons are listed in . For protein structure analyses, protein sequences were retrieved from SGD. PDB files were retrieved from the AlphaFold protein structure database ( ) and visualized using UCSF ChimeraX version: 1.6.1 ( ). Fluorescence microscopy and image analyses. Cells expressing GFP-tagged proteins were grown until mid-log phase and either left untreated or exposed to As(III) and/or 0.2 mg/ml CHX added to the culture at the same time as the As(III). To induce nuclear export of Mig1-GFP, cells were first grown in glucose-containing medium and then shifted to medium containing 2% glycerol. Where indicated, cells were pretreated with 1.5 mM As(III) for 1 h. All samples were fixed in 3.7% formaldehyde (RT, 30 min) followed by two washes in 1x PBS. Nuclear staining was done by incubating fixed cells in ethanol (RT, 40 min), washing in 1x PBS, and resuspending in 4’,6-diamidino-2-phenylindole (DAPI) solution (D1306; Thermo Fisher Scientific). Fluorescent signals were detected using a Zeiss Axiovert 200 M (Carl Zeiss Microscopy, München, Germany) fluorescence microscope equipped with Plan-Apochromat 1.40 objectives and appropriate fluorescence light filter sets. Images were taken with a digital camera (AxioCamMR3). The ZEISS ZEN PRO software (Carl Zeiss Microscopy) was used to capture the images and the ImageJ-Fiji software ( ) for quantifications. The steady-state localization of nuclear transport reporters (GFP-tcNLS, Nab2NLS-GFP, Pho4NLS-GFP, GFP) was determined largely as described previously ( ; ). Cells were grown to mid-log phase in YNB medium containing 2% raffinose and then expression of the reporters was induced by the addition of 0.5% galactose for 4 h followed by the addition of 1.5 mM As(III) for 1 h. N/C ratios were quantified by measuring the mean fluorescence intensity in the nucleus and in the cytosol. The nucleus was outlined along the NE using Nup49-mCherry. Care was taken to exclude the vacuole when choosing a field in the cytosol. All measured values were corrected for background fluorescence and the ratio of nuclear versus cytosolic signal (N/C ratio) was calculated for three replicates and averaged. Protein aggregate isolation and Western blotting. Insoluble protein aggregates were isolated as described previously ( ; ). Cells were grown to mid-log phase, unexposed or exposed to 1.5 mM As(III), collected by centrifugation, resuspended in lysis buffer (50 mM potassium phosphate buffer pH 7, 1 mM EDTA, 5% glycerol, 1 mM PMSF, EDTA-free protease inhibitor cocktail from Roche Diagnostics, Basel, Switzerland), and lysed with 2.5 mg/ml lyticase (Sigma-Aldrich: 30 min, 30°C). Cells were disrupted using sonication on ice (Sonifier 150, Branson Ultrasonics, Danbury, CT, USA; 8× 5 s pulses, 50% amplitude) and the total lysates collected by centrifugation. Protein concentrations in the lysates were adjusted to equal for all samples. Aggregated proteins were isolated by centrifugation of the protein lysates, resuspended in lysis buffer containing 20% NP40 twice, followed by washing and resuspension of the pellet in lysis buffer. The final resuspension was aided by sonication (2×5 sec, 50% amplitude on ice). 5X SDS loading buffer (4% SDS, 250 mM Tris Buffer pH 6.8, 16% β-mercaptoethanol, 30% glycerol, bromophenol blue) was added to all samples followed by boiling for 5 min at 95°C. Proteins were separated on a Criterion 4–20% stain free precast gel (Bio-Rad Laboratories, Hercules, CA, USA) and visualized using a ChemiDoc XRS+ system with UV-activation (Bio-Rad). For Western blot analysis, proteins were transferred to a PVDF membrane using TransBlot Turbo transfer system (semi-dry transfer, Bio-Rad). Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) for 1 h at RT followed by over-night incubation at 4°C with an anti-GFP antibody (1:8000, A11122; Invitrogen, Waltham, MA, USA). Membranes were washed 3X with TBS-T, incubated for 2 h with anti-rabbit-IgG secondary antibody (1:5000, 84546; Invitrogen, Waltham, MA, USA) and washed with TBS-T followed by signal detection using the ChemiDoc system. Immuno-electron microscopy. Immuno-EM was performed largely as described previously ( ). Cells were grown to early log phase and either left untreated or exposed to 1.5 mM As(III) for 1 h. Samples were high-pressure frozen (Wohlwend HPF Compact 3, Sennwald, Switzerland) followed by freeze substitution in 2% uranyl acetate (UA) dissolved in acetone, and embedded into HM20 lowicryl resin (Polysciences) that was UV polymerised at −50°C. The resin was sectioned in 70 nm thin sections and placed on mesh grids. The sections were fixed in 1% paraformaldehyde in PBS for 10 min and blocked with 0.1% fish skin gelatine and 0.8% BSA in PBS for 1 h. For detection of Nups, samples were incubated for 2 h with 1:120 dilution of the mouse monoclonal anti-NPC antibody Mab414 (Abcam ab24609) at 4°C, followed by incubations at room temperature with 1:150 dilution of rabbit anti-mouse immunoglobulin (Agilent/Dako, E0433) for 1 h and with 1:70 diluted 10 nm gold-conjugated protein A antibody (CMC UMC Utrecht, The Netherlands) for 30 min. Glutaraldehyde (2.5%) was applied to sections for 1 h, followed by contrast staining in 2% UA for 5 min and 1 min in Reynold’s lead citrate ( ). Three washing steps (20 min, PBS) were carried out after incubations with each antibody. Images were acquired at 120 kV on a Tecnai T12 transmission electron microscope equipped with a Ceta CMOS 16M camera (Thermofischer scientific, Eindhoven, the Netherlands). Quantifications were made using IMOD ( ) and statistics with GraphPad Prism 10. 

All yeast strains and plasmids used in this work are listed in . The S. cerevisiae strains are based on BY4741 ( ), the deletion collection ( ), the collection of temperature-sensitive mutants of essential yeast genes ( ), and the GFP collection ( ). The strains harbouring nuclear transport reporters have been described previously ( ). Plasmids containing HA-tagged versions of Kap123, Sxm1, Nup84, and Nup188 were constructed via Gateway Recombination Cloning (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Gene sequences were amplified by PCR using genomic DNA as template, inserted into the donor plasmid pDONR221 (Thermo Fisher Scientific, Waltham, MA, USA) and then into the destination vector pAG426GPD-ccdB-HA (Addgene plasmid #14252; http://n2t.net/addgene:14252 ) ( ). All plasmids were verified by sequencing. Cells were routinely grown at 30°C in minimal YNB (yeast nitrogen base) medium with 2% glucose as carbon source. Cells containing nuclear transport reporters were grown with 2% raffinose as carbon source and protein expression was induced with 0.5% galactose for 4 h. For growth assays on plates, cells were grown until log phase, the optical density (OD) at 600 nm adjusted to equal for all cultures, diluted in 10-fold steps, plated using a sterilized stamp, and incubated for 2–3 days at 30°C. Where indicated, the following chemicals were added: sodium arsenite (NaAsO 2 , S7400), cycloheximide (C7698) (both from Sigma-Aldrich, St. Louis, MO, USA), biotinyl p-aminophenyl arsenic acid (As-biotin, B394970; Toronto Research Chemicals, North York, Canada), and monomethylarsonous acid (CH 5 AsO 2 , M565100; LGC Standards, Wesel, Germany). 

Proteome-wide identification of arsenic-binding proteins was performed with mtq2Δ cells grown to early log phase in YNB medium. For the last hour, the medium was switched to YEPD (Yeast Extract Peptone Dextrose) with 2% glucose and split into control and treatment cultures. The cultures were pretreated for 10 min with either As(III) or MAs(III) as blocking agents, followed by 10 min incubation with As-biotin at 30°C. The cultures were collected, pelleted, and frozen at −80°C or lysed directly. Cell lysis was performed by bead beating (1 min, 4°C) in IP buffer (1x TNT buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton, pH 7–7.5)), 1x protease inhibitor (Complete mini, EDTA-free; Roche), 1x phosphatase inhibitor (PhosSTOP Easypack; Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Streptavidin agarose beads (ThermoFisher, 20353) were prepared by a 1x TNT buffer wash and aliquoted to all lysates. After a 1 h incubation (4°C, rotating), pull-down was performed by centrifugation (1000× g , 1 min) and washing three times in 1x TNT. Proteins present in the pull-down were separated by SDS-PAGE electrophoresis followed by identification using microcapillary liquid chromatograph-tandem mass spectrometry (LC/MS/MS) at the Taplin Mass Spectrometry Facility at Harvard Medical School. To validate arsenic-binding to selected proteins, we used wild type cells carrying plasmids with HA-tagged versions of the corresponding genes or cells that harboured GFP-tagged versions of the genes in their genomes. After As-biotin pull-down and SDS-PAGE as described above, the proteins were visualized by Western blotting using anti-GFP rabbit IgG (1:8000, A11122; Invitrogen, Waltham, MA, USA) or anti-HA mouse IgG (1:1000, sc-7392; Santa Cruz Biotechnologies, Dallas, TX, USA) primary antibodies, and anti-rabbit IgG (1:3000, 84564) and anti-mouse IgG (1 : 5000, 84545) secondary antibodies (both from Invitrogen (Waltham, MA, USA)). A more extensive description of the Western blot protocol is provided in the section ‘ ’ below. 

Negative genetic interactors (including negative genetic, synthetic growth defect, synthetic lethality) of selected arsenic-binding hits were retrieved from the Saccharomyces genome database (SGD) ( ) and compared to a compendium of 712 As(III) sensitive S. cerevisiae mutants that contains the genes identified at least once in four previous genome-wide phenotypic screens ( ; ; ; ). The significance of the overlaps between datasets was calculated by the hyper-geometric test. All datasets used for comparisons are listed in . For protein structure analyses, protein sequences were retrieved from SGD. PDB files were retrieved from the AlphaFold protein structure database ( ) and visualized using UCSF ChimeraX version: 1.6.1 ( ). 

Cells expressing GFP-tagged proteins were grown until mid-log phase and either left untreated or exposed to As(III) and/or 0.2 mg/ml CHX added to the culture at the same time as the As(III). To induce nuclear export of Mig1-GFP, cells were first grown in glucose-containing medium and then shifted to medium containing 2% glycerol. Where indicated, cells were pretreated with 1.5 mM As(III) for 1 h. All samples were fixed in 3.7% formaldehyde (RT, 30 min) followed by two washes in 1x PBS. Nuclear staining was done by incubating fixed cells in ethanol (RT, 40 min), washing in 1x PBS, and resuspending in 4’,6-diamidino-2-phenylindole (DAPI) solution (D1306; Thermo Fisher Scientific). Fluorescent signals were detected using a Zeiss Axiovert 200 M (Carl Zeiss Microscopy, München, Germany) fluorescence microscope equipped with Plan-Apochromat 1.40 objectives and appropriate fluorescence light filter sets. Images were taken with a digital camera (AxioCamMR3). The ZEISS ZEN PRO software (Carl Zeiss Microscopy) was used to capture the images and the ImageJ-Fiji software ( ) for quantifications. The steady-state localization of nuclear transport reporters (GFP-tcNLS, Nab2NLS-GFP, Pho4NLS-GFP, GFP) was determined largely as described previously ( ; ). Cells were grown to mid-log phase in YNB medium containing 2% raffinose and then expression of the reporters was induced by the addition of 0.5% galactose for 4 h followed by the addition of 1.5 mM As(III) for 1 h. N/C ratios were quantified by measuring the mean fluorescence intensity in the nucleus and in the cytosol. The nucleus was outlined along the NE using Nup49-mCherry. Care was taken to exclude the vacuole when choosing a field in the cytosol. All measured values were corrected for background fluorescence and the ratio of nuclear versus cytosolic signal (N/C ratio) was calculated for three replicates and averaged. 

Insoluble protein aggregates were isolated as described previously ( ; ). Cells were grown to mid-log phase, unexposed or exposed to 1.5 mM As(III), collected by centrifugation, resuspended in lysis buffer (50 mM potassium phosphate buffer pH 7, 1 mM EDTA, 5% glycerol, 1 mM PMSF, EDTA-free protease inhibitor cocktail from Roche Diagnostics, Basel, Switzerland), and lysed with 2.5 mg/ml lyticase (Sigma-Aldrich: 30 min, 30°C). Cells were disrupted using sonication on ice (Sonifier 150, Branson Ultrasonics, Danbury, CT, USA; 8× 5 s pulses, 50% amplitude) and the total lysates collected by centrifugation. Protein concentrations in the lysates were adjusted to equal for all samples. Aggregated proteins were isolated by centrifugation of the protein lysates, resuspended in lysis buffer containing 20% NP40 twice, followed by washing and resuspension of the pellet in lysis buffer. The final resuspension was aided by sonication (2×5 sec, 50% amplitude on ice). 5X SDS loading buffer (4% SDS, 250 mM Tris Buffer pH 6.8, 16% β-mercaptoethanol, 30% glycerol, bromophenol blue) was added to all samples followed by boiling for 5 min at 95°C. Proteins were separated on a Criterion 4–20% stain free precast gel (Bio-Rad Laboratories, Hercules, CA, USA) and visualized using a ChemiDoc XRS+ system with UV-activation (Bio-Rad). For Western blot analysis, proteins were transferred to a PVDF membrane using TransBlot Turbo transfer system (semi-dry transfer, Bio-Rad). Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) for 1 h at RT followed by over-night incubation at 4°C with an anti-GFP antibody (1:8000, A11122; Invitrogen, Waltham, MA, USA). Membranes were washed 3X with TBS-T, incubated for 2 h with anti-rabbit-IgG secondary antibody (1:5000, 84546; Invitrogen, Waltham, MA, USA) and washed with TBS-T followed by signal detection using the ChemiDoc system. 

Immuno-EM was performed largely as described previously ( ). Cells were grown to early log phase and either left untreated or exposed to 1.5 mM As(III) for 1 h. Samples were high-pressure frozen (Wohlwend HPF Compact 3, Sennwald, Switzerland) followed by freeze substitution in 2% uranyl acetate (UA) dissolved in acetone, and embedded into HM20 lowicryl resin (Polysciences) that was UV polymerised at −50°C. The resin was sectioned in 70 nm thin sections and placed on mesh grids. The sections were fixed in 1% paraformaldehyde in PBS for 10 min and blocked with 0.1% fish skin gelatine and 0.8% BSA in PBS for 1 h. For detection of Nups, samples were incubated for 2 h with 1:120 dilution of the mouse monoclonal anti-NPC antibody Mab414 (Abcam ab24609) at 4°C, followed by incubations at room temperature with 1:150 dilution of rabbit anti-mouse immunoglobulin (Agilent/Dako, E0433) for 1 h and with 1:70 diluted 10 nm gold-conjugated protein A antibody (CMC UMC Utrecht, The Netherlands) for 30 min. Glutaraldehyde (2.5%) was applied to sections for 1 h, followed by contrast staining in 2% UA for 5 min and 1 min in Reynold’s lead citrate ( ). Three washing steps (20 min, PBS) were carried out after incubations with each antibody. Images were acquired at 120 kV on a Tecnai T12 transmission electron microscope equipped with a Ceta CMOS 16M camera (Thermofischer scientific, Eindhoven, the Netherlands). Quantifications were made using IMOD ( ) and statistics with GraphPad Prism 10. 

1 

