Molecular Microbiology (2015) 98(1), 162–174 ■
doi:10.1111/mmi.13113 First published online 22 July 2015
Identiﬁcation of critical residues for transport activity of Acr3p, the Saccharomyces cerevisiae As(III)/H+ antiporter Katarzyna Markowska, Ewa Maciaszczyk-Dziubinska, Magdalena Migocka, Donata Wawrzycka and Robert Wysocki* Institute of Experimental Biology, University of Wroclaw, 50-328 Wroclaw, Poland.
Summary 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.
Introduction The highly toxic and carcinogenic inorganic arsenic species arsenite (As(III)/As(OH)3) and arsenate (As(V)/ AsO43−) are widely present in the environment from both Accepted 26 June, 2015. *For correspondence. E-mail robert [email protected]
; Tel. (+48) 71 375 4126; Fax (+48) 71 375 4118.
© 2015 John Wiley & Sons Ltd
natural and anthropogenic sources. Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water and crops (Nordstrom, 2002; Tapio and Grosche, 2006; Sharma et al., 2014). On the other hand, arsenic is used in modern chemotherapy against acute promyelocytic leukemia (Dilda and Hogg, 2007). Understanding of molecular mechanisms of arsenic detoxification and tolerance is one of the prerequisites to develop transgenic organisms capable of cleaning up arsenic-polluted areas or crops showing minimal arsenic accumulation as well as to cope with metalloid resistance emerging during therapy. The Acr3 family of arsenite transporters represents the most common pathway conferring high-level resistance to toxic metalloids (Rosen and Tamás, 2010; MaciaszczykDziubinska et al., 2012). Members of the Acr3 family are present in bacteria, archaea, unicellular eukaryotes, fungi and lower plants but have been lost in flowering plants and animals (Fu et al., 2009; Indriolo et al., 2010; MaciaszczykDziubinska et al., 2014). However, yeast or fern Acr3 can be successfully expressed in rice and Arabidopsis thaliana, leading to increased tolerance to arsenicals (Ali et al., 2012; Duan et al., 2012; Chen et al., 2013). The Acr3 transporters exhibit ten-transmembrane span topology (Aaltonen and Silow, 2008; Fu et al., 2009) and are similar to Na+-dependent bile acid transporters (Mansour et al., 2007). In microbial species, Acr3 proteins are localized to the plasma membrane to mediate As(III) extrusion out of the cells (Wysocki et al., 1997; Sato and Kobayashi, 1998; Ghosh et al., 1999; López-Maury et al., 2003; Ordóñez et al., 2005; Fu et al., 2009). The Acr3p transporter from S. cerevisiae (Maciaszczyk-Dziubinska et al., 2010) and probably that from the cyanobacterium Synechocystis also transport antimonite (Sb(III)) (López-Maury et al., 2003), while others seem to be specific for As(III) (Sato and Kobayashi, 1998; Fu et al., 2009; Villadangos et al., 2012). Surprisingly, the Shewanella oneidensis Acr3 confers resistance only to As(V) when expressed in Escherichia coli and does not bind As(III) (Xia et al., 2008). This suggests that Acr3 from Shewanella oneidensis might be specific for As(V). In some microorganisms, such as the actinobacterium Mycobacterium tuberculosis or social amoeba Polysphondylium pallidum, the C-terminal end of Acr3 is fused with the arsenate reductase domain, which probably enables coupling of As(V) reduction to As(III) and
Mutagenesis of Acr3 transmembrane segments 163
its immediate export out of the cell (Wu et al., 2010). Importantly, the presence of Acr3 in the fern Pteris vittata is the base of its arsenic hyperaccumulating properties by sequestering As(III) into the vacuolar lumen (Indriolo et al., 2010). However, the fern Acr3 expressed in A. thaliana localizes to the plasma membrane, contributing to As(III) efflux into the external medium, xylem or apoplast (Chen et al., 2013). Similarly, the yeast Acr3p is also sorted to the plasma membrane when expressed in rice (Duan et al., 2012). Thus, subcellular localization of Acr3 proteins from lower plants deserves further studies. Recently, we found that the yeast Acr3p catalyzes low affinity As(III)/H+ and Sb(III)/H+ antiport coupled to the proton-motive force; however, compared with As(III), the rate of Sb(III) transport was much slower (MaciaszczykDziubinska et al., 2011). This finding is in good agreement with the observation that the yeast Acr3p mediates lowlevel resistance to Sb(III) (Maciaszczyk-Dziubinska et al., 2010). The Corynebacterium glutamicum Acr3 has also been shown to act as an As(III)/H+ antiporter, strongly suggesting that the mechanism of As(III) transport is conserved among members of the Acr3 family (Villadangos et al., 2012). Moreover, a highly conserved cysteine residue (Cys129 in C. glutamicum, Cys138 in Alkaliphilus metalliredigens, Cys151 in S. cerevisiae) located in the middle of the fourth transmembrane segment (TM4) of Acr3p is crucial for transport activity (Fu et al., 2009; Villadangos et al., 2012; Maciaszczyk-Dziubinska et al., 2014). It has been hypothesized that a low-affinity interaction of As(III) with a single thiol group in Acr3 is necessary for the translocation process (Fu et al., 2009; Villadangos et al., 2012). Importantly, replacement of an equivalent cysteine residue with alanine in the S. oneidensis Acr3 did not alter its ability to confer resistance to As(V) (Xia et al., 2008). Additionally, we have demonstrated that mutation of Cys90 and Cys169, which are located in predicted cytosolic loops of the S. cerevisiae Acr3p, resulted in failure to exit the ER and decreased transport activity respectively (Maciaszczyk-Dziubinska et al., 2014). Mutagenesis analysis of several hydrophilic residues located in the predicted transmembrane helices of C. glutamicum Acr3 revealed that another conserved residue, glutamate (Glu305) in TM9, is also required for the As(III)/H+ exchange (Villadangos et al., 2012). In this study, to gain better insight into the mechanism of As(III)/H+ antiport, we performed a mutagenesis study of 26 conserved hydrophilic and aromatic residues predicted to localize in hydrophobic helices of the yeast Acr3p. As a result of this analysis, we identified several residues that are required for proper structure and sorting of Acr3p as well as for transport activity. Based on the topological similarities to the bile acid sodium symporter ASBT, we propose a speculative model of the As(III)/H+ antiport mechanism. © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
Results Construction of acr3 alleles Following our previous characterization of the yeast Acr3p (Maciaszczyk-Dziubinska et al., 2014), we aimed to identify additional residues that are engaged in the As(III)/H+ exchange. We hypothesized that conserved hydrophilic residues located in putative transmembrane regions of the yeast Acr3p might be involved in the As(III)/H+ antiport. It has been shown that charged and polar residues are critical for the activity of the sodium/proton antiporters (Hunte et al., 2005; Lee et al., 2013) and the sodiumdependent bile acid symporters of the bile/arsenite/ riboflavin transporter superfamily (Hu et al., 2011; Zhou et al., 2014). Based on the alignment of Acr3 protein sequences from distantly related organisms (Fig. 1A), we chose a number of conserved residues for a site-directed mutagenesis, including 5 charged (Asp34, Arg150, Arg230, Glu353, Glu380) and 14 polar residues (Ser74, Ser113, Asn117, Asn176, Ser177, Gln180, Tyr260, Thr261, Tyr290, Thr346, Ser349, Asn350, Asn351, Thr374) located in the putative transmembrane segments (Fig. 1B). In addition, we included seven highly conserved nonpolar aromatic residues (Trp118, Trp130, Trp158, Phe266, Phe291, Phe345, Phe352) in our study (Fig. 1) as aromatic rings may be important for protein-ligand recognition, establishment of protein structure or the substrate translocation pathway (Kuwabara et al., 2004; Nevo, 2007; Salonen et al., 2011). The selected residues were replaced with alanines by site-directed mutagenesis using the centromeric plasmid containing the ACR3 gene fused to GFP (pACR3-GFP) as a template. Next, the yeast acr3 deletion mutant (acr3Δ) was used as a host strain for transformation with plasmids expressing 26 Acr3 mutant variants together with the wild-type ACR3 plasmid and an empty vector as a control followed by phenotypic characterization. Functional characterization of Acr3 mutants To test the functionality of constructed Acr3 mutant variants, the acr3Δ transformants were spotted on plates containing various concentrations of As(III) (Fig. 2). Expression of 10 mutants (D34A, S113A, W118A, S177A, Q180A, Y260A, T261A, T346A, N350A, T374A) conferred high-level resistance to As(III) and they were classified as functional (Table 1). Consistently, these mutants showed no defect in As(III) extrusion from yeast cells (Fig. 3). The second group of mutants (S74A, N117A, W130A, N176A, F291A) exhibited slight or moderate complementation of As(III) hypersensitivity of acr3Δ and reduced As(III) efflux (Figs 2 and 3); these five variants were categorized as partially functional (Table 1). Finally, 11 Acr3 mutant proteins (R150A, W158A, R230A, F266A, Y290A, F345A, S349A, N351A, F352A, E353A, E380A) appeared to be
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Table 1. Summary of functional analysis of Acr3 mutant proteins. Mutant name
Total protein level
PM protein level
D34A S74A S113A N117A W118A W130A R150A W158A N176A S177A Q180A R230A Y260A T261A F266A Y290A F291A F345A T346A S349A N350A N351A F352A E353A T374A E380A
TM1 TM2 TM3 TM3 TM3 TM3 TM4 TM4 TM5 TM5 TM5 TM6 TM7 TM7 TM7 TM8 TM8 TM9 TM9 TM9 TM9 TM9 TM9 TM9 TM10 TM10
+++ ++ +++ ++ +++ + − − + +++ +++ − +++ +++ + /− − ++ + /− +++ − +++ − − − +++ −
+++ ++ +++ + +++ + − − + +++ +++ − +++ +++ − − ++ − +++ − +++ − − − +++ −
N N N N N N D D D N N D N N N D N D N N N D N N N N
PM PM,ER PM ER,PM PM VA,PM ER VA,PS,PM ER PM PM PM,ER PM,ER PM PM ER PM ER PM PM PM ER PM PM PM PM
ND N ND D ND D R D R ND ND D ND ND N R N R ND N ND R N N ND N
F PF F PF F PF NF NF PF F F NF F F NF NF PF NF F NF F NF NF NF F NF
Mutated region: TM, transmembrane span. As(III) resistance and transport activity: −, none; + or ++, partial; +++, full. Total and plasma membrane (PM) protein level: N, normal; D, decreased; R, residual; ND, not determined. Subcellular localization: ER, endoplasmic reticulum; PM, plasma membrane; PS, punctate structures in the cytoplasm; VA, vacuole. Mutant class: F, functional; PF, partially functional; NF, non-functional.
non-functional as they were unable to confer resistance to As(III) or export As(III) out of the cells (Figs 2 and 3, Table 1). In addition to As(III), Acr3p mediates the transport of Sb(III) (Maciaszczyk-Dziubinska et al., 2010). Consistently, cells expressing non-functional or partially functional Acr3 mutants were also more sensitive to Sb(III) (Fig. 2). None of the constructed mutants exhibited increased resistance to Sb(III) or As(V), suggesting no change in the specificity of Acr3p variants (Fig. 2; data not shown). Protein level and subcellular localization of Acr3 mutant variants Expression of all tested Acr3 mutants was not affected at the mRNA level (data not shown). However, Western
blot analysis of total protein extracts using antibodies against GFP-fused Acr3 protein revealed a significant reduction of total protein level in the case of seven Acr3 mutants (R150A, W158A, N176A, R230A, Y290A, F345A, N351A) (Fig. 4A). This suggests that these mutations may be detrimental for proper folding of Acr3p, leading to its perturbed subcellular localization and accelerated degradation. Epifluorescence microscopy was used to examine the subcellular localization of GFP-tagged Acr3 variants. A majority of mutant Acr3 proteins showed a high fluorescence signal at the cell surface similar to the wild-type Acr3-GFP, indicative of proper localization in the plasma membrane (Fig. 4B). Consistent with the Western blot data, mutants with a low protein level also displayed
Fig. 1. Amino acid sequence alignment of representative members of the Acr3 family and topology of the yeast Acr3p transporter. A. Alignment of protein sequences of Acr3 family members was performed with Clustal Omega (Sievers et al., 2011) and included Acr3 transporters from fungi (budding yeast, Saccharomyces cerevisiae, Sce, NCBI accession no. DAA11615), plants (fern, Pteris vittata, Pvi, ADP20955), social amoeba (Polysphondylium pallidum, Ppa, EFA79047), choanoflagellates (Monosiga brevicollis, Mbr, EDQ91878), bacteria (actinobacteria, Corynebacterium glutamicum, Cgl, YP_225795; firmicutes, Bacillus subtilis, Bsu, BAA12433; proteobacteria, Shewanella oneidensis, Son, AAN53615; cyanobacteria, Synechocystis sp. PCC 6803, Syn, BAA18405) and archaea (euryarchaeota, Archaeoglobus fulgidus, Afu, AAB90761; Korarchaeota, Candidatus Korarchaeum cryptofilum, Cku, WP_012308942). The conserved residues subjected to site-directed mutagenesis are highlighted in black. B. Topology of S. cerevisiae Acr3p was predicted by the HMMTOP method (Tusnády and Simon, 2001), which best fits the Acr3 topology determined for bacterial Acr3 proteins (Aaltonen and Silow, 2008; Fu et al., 2009). Amino acid residues chosen for alanine-replacement analysis are in black circles. A−B. Cysteine residues previously shown to be necessary for the Acr3 function are also marked (Maciaszczyk-Dziubinska et al., 2014) © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
Mutagenesis of Acr3 transmembrane segments 165
Sce Pvi Ppa Mbr Cgl Bsu Son Syn Afu Cko
----------------------MSE----DQKSENSVPSKVNMVNRT--DILTTIKSLSWLDLMLPFTIILSIIIAVIISVYVPSSRHTFDAEGHPNLMGVSIPLTVGMIVMMIPPICKVSWES 96 -----MENSSA--ERKQQLALDIADGNDPSD-------AAKNPDGRT--KLQGLFKQLSLLDRYLYVWIFIVMAVSIIFGYYVKGVKKAFQ---VAEITSVSLPIAIGLWVMMYPVLCKVQYEI 105 MQILWLVKFSYVFKWNIFILLDKTRGCDYIYSFSFSAIAVVQYILIN--SYYKMMKGLSLVDKLLPLWILLFMTAGILIGYYVPSVEDAFS---RSTLASVSLPIAVGLLLMMYPVFCRIHYEK 119 ------------MVTKMPAAYDALSETDVSQPSSAAM-PAVSAEAGSSETGSAPPAAIDTFARFLTVWVLLAMAVGVLVGYYVPAIPDALE---SATVAQISIPIAIFIWIMVYPMLLQIDLTA 108 ------------------------------------------MTNST--QTRAKPARISFLDKYIPLWIILAMAFGLFLGRSVSGLSGFLG---AMEVGGISLPIALGLLVMMYPPLAKVRYDK 77 ------------------------------------------------------MKRLSFLDRYLTIWIFLAMALGIGLGFIFPSFVGGLN---KLQVGTTSIPLAIGLVLMMYPPLAKVRYEE 67 ---------------------------------------------------------MGIFERYLSVWVGLSILAGVVLGNVVPNVFAAVA---ALEFAHVNLVIALFIWVMIYPMMVQIDFSA 64 ------------------------------------------MVNRINPKAIKAGGTLNLFEKYLTLWVALCIVIGIALGKLLPAVAQTLD---SWSIYNVSIPIAICLFFMMYPIMVKIDFSQ 79 ----------------------MSR----IDDIERDIKKAEKVVEHY--VTLEEKKELSIIEKFLTLWIFLAIIGGVALGYVYPDIANIIG---SLSIGTTSIPIAIGLILMMYPPLAKVRYEE 93 -----------------------------------------------------MERKLGLIEKYLTIWIFLAMIFGIALGYFYPGISIMLA---GMSVGTTSIPIAAGLILMMYPPLAKVRYEE 68
Sce Pvi Ppa Mbr Cgl Bsu Son Syn Afu Cko
IHKYFYR--SYIRKQLALSLFLNWVIGPLLMTALAWMALF-------------------DYKEYRQGIIMIGVARCIAMVLIWNQIAGGDNDLCVVLVITNSLLQMVLYAPLQIFYCYVISHDH LGGVLRQAGS--LKTISLSVVLNWVVGPALMTGLAWATLP-------------------DLPDFRTGVILVGIARCIAMVLIWNDLAKGDADYCAILVAINSILQIILFTPVALLYLKVVS--LASKFVKDRSFFIRQIGVSLVVNWIVAPLVMVAIAWATLP-------------------DLVHYRSGVILVGVARCIAMVLIWNHLAKGNDEFCALVVAINSVIQMFFYGPLSYFFVVILG--LQRVRYN-----LKPILVTTGINYLIQPFCMYGLAKLFIDVAFEDVIS---------DQLQEEYVTGAVILGGSPCTAMVFVWSALVSGDAAYTLVQVAVNDILIFILYIPTMMLLLQ-----TKQIATD-----KHLMGVSLILNWVVGPALMFALAWLFLP-------------------DQPELRTGLIIVGLARCIAMVLVWSDMSCGDREATAVLVAINSVFQVAMFGALGWFYLQVLPSWL IGRVFKD-----IKVLILSLVQNWIIGPTLMFILAIIFLP-------------------DKPEYMIGLIMIGLARCIAMVIVWNDLSKGDTEYAAGLVAFNSIFQMLFSSVYAYIFVTVIPQWL VKDVGNA-----PKGLILTLFINWLVKPFTMALLGWLFFKLVFADWVD---------PQTASEYIAGMILLGVAPCTAMVFVWSQLTKGDPNYTLVQVSVNDIIMVFLFAPLSALLLG-----ARQAVKA-----PKPVILTLVVNWVIKPFTMVIFAQFFLGYLFAPLLTATEIIRGQEVTLANSYIAGCILLGIAPCTAMVLMWGYLSYSNQGLTLVMVAVNSLAMLFLYAPLGKWLLA-----MGKVFSN-----LKLLAFSLTQNWIVGPVVMFLLAIALLR-------------------DMPEFMIGVILVGLARCIAMVIVWNELAEGDRELAVGLVAFNAIFQILFYAVYIYIFITFALNAL MGKIFKN-----LKLLSFSLAQNWIFGPIVMFLLAITLLR-------------------DLPAFMMGVILVGLARCIAMVIVWNELAEGDRELAVGLVAFNAIFQILFYAVYIYIFITLALTGL
Sce Pvi Ppa Mbr Cgl Bsu Son Syn Afu Cko
Sce Pvi Ppa Mbr Cgl Bsu Son Syn Afu Cko
Extracellular H T F D
R S S P V TM1 Y 53 V S I I V I A I I S L I I F T P L M L D W L S L 29 S K I
A E G E Y H R K P Q Y D TM4 G TM2 N TM3 70 134 143 F I I M L A L M M G I G W V S V L A I A T A L P C R L M L T I A P V G M I G M V V I L W I N V M W F L Q N I M S L L P I A P L A 110 89 I 162 Q G C K G Y F K D N D R K Y T V I S H R T S Y I L W S I K P E S D V V N S T S M NH R M N E K Q 2 E S D N V
N R V T S L
L H D
E E V
I Y V Y C F I L Q A P Y L M V Q L L N S I T V L V
Q F I N N S Y G H G K Y A K E L TM7 R TM6 TM8 TM9 TM10 Q 214 268 277 361 370 S I S I S I A S G G V A I A I V A F F A I I A V T C L G L S L L F F F P I P V F E T L P G L G L N G I L V L L S N H Y I A Y F E I M F G I F V T L R A I C F M I I P A L G Q W L F S T P W I T L T L T M S L F I I A A S I A 238 G 249 F 301 L 337 F 389 I M R K S V D Q A R L S A C E E Y N I R S L L Y E K Y C I P Y C L S S L K Y L K E I I R K S R W N N R R Q R G N S T V W HOOC D
© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
199 205 221 212 177 167 178 192 193 168 320 312 325 317 286 276 273 298 303 278 404 379 434 421 370 346 339 383 370 347
166 K. Markowska et al. ■
Fig. 2. Growth of acr3 mutants in the presence of arsenite and antimonite. Serial dilutions of the indicated acr3Δ transformants were spotted on minimal selective media containing various concentrations of metalloids. Plates were photographed after 3 days at 30°C.
vec WT D34A S74A S113A N117A W118A W130A R150A W158A N176A S177A Q180A R230A Y260A T261A F266A Y290A F291A F345A T346A S349A N350A N351A F352A E353A T374A E380A Control
0.1 mM As(III)
0.5 mM As(III)
1 mM As(III)
impaired subcellular localization. Six of these mutant proteins (R150A, N176A, R230A, Y290A, F345A, N351A) localized both near the cell surface and in a ring-shaped structure around the nucleus, which is a typical pattern for proteins residing in the endoplasmic reticulum (ER) (Lowe and Barr, 2007; Merhi et al., 2011; Young et al., 2013). In contrast, the W158A mutant accumulated in the vacuole or cytoplasmic punctate structures that may correspond to endosomes or the Golgi (Lowe and Barr, 2007; Hachiro et al., 2013). Vacuolar accumulation of GFP signal was also apparent for the W130A mutant, while S74A, N117A and Y260A mutants showed the ER signal despite the wild-type total protein levels (Fig. 4B). Having established sorting defects and the decreased stability of some Acr3 mutants, we determined the plasma
10 mM Sb(III)
membrane protein level of partially functional or nonfunctional Acr3 variants to pinpoint mutations affecting the antiport activity solely. We prepared the plasma membrane-enriched fractions from the transformants expressing Acr3 variants followed by Western blot analysis and found that R150A, N176A, Y290A, F345A and N351A were present at residual levels in the plasma membrane fraction, supporting the microscopy data indicating that they were trapped in the ER (Fig. 4C). Four Acr3 mutants showed significantly decreased presence at the plasma membrane, indicating that these mutant proteins exhibit an improper overall structure resulting in either exit delay from the ER (N117A, W130A, R230A) or downregulation via the endocytic pathway (W158A). The remaining Acr3 mutants classified as partially functional (S74A, F291A) or non© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
Mutagenesis of Acr3 transmembrane segments 167
Intracellular As(III) relative levels
Fig. 3. Effect of mutations in putative transmembrane helices of Acr3p on As(III) efflux. Cells were incubated in the presence of 0.1 mM As(III) for 2 h and then exposed to 1 mM As(III) for 2 h. Next, cultures were washed and resuspended in fresh media without As(III). Arsenic content in the indicated acr3Δ transformants was determined at 0 (set to 1) and 4 h after release. Error bars indicate the standard deviations of three assays with duplicate technical replicates each time (n = 3).
functional (F266A, S349A, F352A, E353A, E380A) were present at the wild-type level at the plasma membrane, suggesting that these residues are important for the As(III)/H+ exchange mechanism. Analysis of As(III)/H+ antiport activity of Acr3 mutant proteins To examine the effect of mutations in putative transmembrane segments on the activity of Acr3p in a direct way, we measured the As(III)/H+ antiport activity in inside–out membrane vesicles isolated from the acr3Δ transformants expressing the Acr3 variants which were shown to be present in the plasma membrane (Fig. 4C) but defective in conferring As(III) resistance (Figs. 2 and 3). The rate of gradient dissipation in ΔpH energized vesicles was measured following the addition of 10 mM As(III) (Fig. 5). The S74A and F291A mutants, partially functional in growth and As(III) efflux assays (Figs. 2 and 3), showed almost wildtype antiport activity, excluding a direct role of these residues in the Acr3p mechanism. N117A and W130A mutations resulted in a 30–40% decrease in the As(III)/H+ exchange, but it could be explained by a reduced protein level at the plasma membrane due to sorting defects (Fig. 4). In contrast, two other mutants, W158A and R230A, which showed trafficking problems and decreased protein levels at the plasma membrane similar to N117A and W130A, failed to mediate As(III)-induced translocation of protons. Thus, highly conserved Trp158 in TM4 and Arg230 in TM6 may be critical for maintaining the structure © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
of an active antiporter. On the other hand, despite a proper protein level and subcellular localization, the Ser349A mutation in TM9 led to over 80% reduction of the antiport activity, while F266A in TM7, F352A and E353A in TM9, and E380A in TM10 completely abolished the As(III)/H+ exchange in vesicles. This confirms that Phe266, Ser349, Phe352, Glu353 and Glu380 are involved in the transport mechanism of Acr3p.
Role of Glu353 and Glu380 in transport activity of Acr3p Glu353 and Glu380 residues were chosen for further study because they are hypothesized to serve as proton binding sites. Both glutamate residues were replaced with a negative residue of a shorter side-chain (E353D, E380D), a positively charged residue (E353K, E380K), a non-protonatable residue of similar size (E353Q, E380Q), or a similar volume but hydrophobic residue (E353L, E380L), and the phenotype of resulting mutants was compared with wild-type and alanine-replacement mutants (E353A, E380A). All Glu353 variants did not support the growth of the acr3Δ mutant in the presence of As(III) (Fig. 6A), although they were properly expressed and localized to the plasma membrane (Fig. 6B and C). The Glu353 mutants also failed to mediate the As(III)/H+ antiport (Fig. 6D). These results strongly indicate that the Glu353 residue is essential for transport activity of Acr3p and may contribute to substrate binding. Except for the E380D mutation, the Glu380 variants also appeared to be non-functional (Fig. 6). The E380D mutant partially com-
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A Acr3p-GFP Pho85p Cdc28p Total protein extracts
C Acr3p-GFP Pma1p
Plasma membrane fractions
Fig. 4. Expression and subcellular localization of Acr3 mutant proteins. A. Western blot analysis of total protein extracts prepared from the acr3Δ cells expressing wild-type and mutant variants of Acr3-GFP after 4 h of incubation in the presence of 0.1 mM As(III). Blots were probed with the anti-GFP antibody to detect Acr3-GFP and the anti-PSTAIRE antibody for detection of Cdc28 and Pho85 as a loading control. B. Samples from cultures analyzed above were also examined by fluorescence microscopy to visualize subcellular localization of Acr3 mutant proteins. C. Immunodetection of Acr3-GFP in the plasma membrane-enriched fraction isolated from the indicated acr3Δ transformants. Blots were probed with the anti-GFP antibody to detect Acr3-GFP and the anti-Pma1 antibody as a loading and plasma membrane quality control. © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
Mutagenesis of Acr3 transmembrane segments 169
Glu380 is necessary for proper folding of S. cerevisiae Acr3p as well as the translocation process.
MgATP WT S74A F291A
VO4 (III) S349A
As(III) vec, W158A R230A, F266A, F352A, E353A, E380A
0.01 60 s
Proton gradient dissipation (%)
Fig. 5. As(III)/H+ exchange activity in the everted membrane vesicles of the acr3Δ transformants expressing wild-type and mutant variants of Acr3p. A. As(III)-dependent proton movements were monitored by following the changes in acridine orange absorbance as described under ‘Experimental Procedures’. Vesicle acidification was initiated by the addition of 2 mM of ATP (first arrow). Next, 0.5 mM of sodium orthovanadate was added to inhibit H+-ATPase and to maintain a steady-state acidic-inside pH gradient (second arrow). At the indicated time point, 10 mM of As(III) was added to initiate metalloid-dependent proton movement, resulting in acidification of the environment and absorbance recovery (third arrow). Traces are representative of three independent assays. B. As(III)-induced proton gradient dissipation in the indicated transformants. Error bars indicate the standard deviations of three experiments with duplicate technical replicates each time (n = 3).
plemented As(III) sensitivity of the acr3Δ strain (Fig. 6A) and showed decreased antiport activity (Fig. 6D), probably due to trafficking problems resulting in a reduced protein level at the plasma membrane (Fig. 6B and C). Note that although replacement of Glu380 with Leu also caused retention of this mutant protein in the ER, no As(III)/H+ exchange was observed in the vesicles isolated from the E380L mutant (Fig. 6B and C). We speculate that © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
In this study, we identified several charged/polar and aromatic residues localized in the predicted transmembrane segments of the yeast arsenite transporter Acr3p that are important for As(III)/H+ antiport activity. A similar mutational analysis of bacterial Acr3 from C. glutamicum revealed two residues, Cys129 in TM4 and Glu305 in TM9, as necessary for the As(III) efflux (Villadangos et al., 2012). Notably, in the yeast Acr3p, these residues correspond to Cys151 and Glu353 (Fig. 1A), which are also important for As(III)/H+ exchange activity (Figs. 3 and 5) (Maciaszczyk-Dziubinska et al., 2014). Interestingly, three mutations (S349A, F352A, E353A) that impaired transport activity of S. cerevisiae Acr3p (Figs 3 and 5) are clustered in the middle of TM9 (Fig. 1B). This region is highly conserved in the Acr3 family, and it is tempting to speculate that residues located in the middle of TM9 might contribute to an As(III)-binding site. Consistently, the presence of aromatic and negatively charged residues is a common structural feature of the substrate binding site in secondary active transporters (Shi, 2013). The bulky side chains of aromatic residues are important for both binding the substrate and the formation of occluded conformation (see discussion later), while negatively charged residues are involved in the coordination of ions. Glu353 (TM9) and possibly Glu380 (TM10) of the yeast Acr3p might be subjected to a process of protonation and deprotonation required for substrate binding and transport, as is commonly observed in H+-coupled secondary active transporters (Hunte et al., 2005; Reyes et al., 2009; Dang et al., 2010; Lee et al., 2013; Kaback, 2015). In support of this hypothesis, Glu353 in the yeast Acr3p (Fig. 6) and its equivalent residue Glu305 in the C. glutamicum Acr3 (Villadangos et al., 2012) are essential for the As(III)/H+ antiport. Replacement of Glu380 with Ala, Leu, Gln and Lys resulted in a loss of antiport function. Also, the E380L mutation led to the retention of Acr3p in the ER (Fig. 6B and C). In contrast, despite similar defects in trafficking to the plasma membrane, the E380D mutation mildly affected transport activity (Fig. 6B and C). Thus, a negatively charged residue in the middle of TM10 is necessary for transport activity of the yeast Acr3p and might serve as a second proton binding site. Surprisingly, mutation of a residue equivalent to Glu380 in the C. glutamicum Acr3 did not significantly impair the As(III)/H+ antiport (Villadangos et al., 2012); thus, it would be interesting to study the importance of this glutamate residue for transport activity of Acr3 proteins from other representative organisms.
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E3 53 E3 D 53 E3 K 53 E3 Q 53 E3 L 53 A E3 80 E3 D 80 K E3 80 E3 Q 8 E3 0L 80 A
Plasma membrane fractions
E380Q E380L E380A Control
1 mM As(III)
0.1 mM As(III) 0.5 mM As(III)
VO 4(III) As(III)
As(III) E353D vec E353K, E353Q E353L, E353A
0.01 60 s
vec E380K, E380Q E380L, E380A
Fig. 6. Functional analysis of E353 and E380 variants of Acr3p. A. Growth of acr3Δ cells transformed with plasmids expressing indicated mutants of Acr3p-GFP on minimal medium in the presence of As(III). Plates were incubated at 30°C and photographed after 3 days. B. Subcellular localization of Acr3p-GFP mutants was analyzed by fluorescence microscopy. Live cells were examined after 4 h of incubation in the presence of 0.1 mM of As(III). C. The level of mutant Acr3 proteins in the plasma membrane fraction was analyzed by immunoblotting with the anti-GFP antibody to detect Acr3p-GFP and the anti-Pma1 antibody as a control. D. As(III)-driven proton movement in the everted membrane vesicles from wild-type and Acr3p mutants. Experiments were performed as described in Fig. 5 and under ‘Experimental procedures’.
There are no structural data for Acr3 proteins that could help to understand the mechanism of the As(III)/H+ antiport. However, the crystal structures of two bacterial homologs of the bile acid sodium symporter ASBT, which share distant sequence homology with Acr3p (Fig. 7)
(Mansour et al., 2007), have recently been determined (Hu et al., 2011; Zhou et al., 2014). ASBTs from Neisseria meningitidis (ASBTNm) and Yersinia frederiksenii (ASBTYf) consist of 10 transmembrane segments that form two large domains, a core domain of six helices (TM3–TM5 © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
Mutagenesis of Acr3 transmembrane segments 171 ACR3 MSEDQKSENSVPSKVNMVNRTDILTTIKSLSWLDLMLPFTIILSIIIAVIISVYVPSSRHTFDAEGHP 68 ASBT ----------------------------MLVKITRLFPVWALLLSVAAYFRPTTFT------------ 28
TM1 ACR3 NLMGVSIPLTVGMIVMMIPPICKVSWESIHKYFYRSYIRKQLALSLFLNWVIGPLLMTALAWMALFDY 136 ASBT GIGPYVGPLLMLIMFAMGVTLRLDDFKRVLSR------PAPVAAATFLHYLIMPLTAWILAMLF-RMP 89
ACR3 KEYRQGIIMIGVARCIAMVLIWNQIAGGDNDLCVVLVITNSLLQMVLYAPLQIFYCYVISHDHLNTSN 204 ASBT PDLSAGMVLVGSVASGTASNVMIYLAKGD----VALSVTISAVSTLVGVFATPLLTRLYVDA----TI 149
Fig. 7. Comparison of membrane topology and sequence alignment of As(III)/H+ antiporter Acr3p from S. cerevisiae and Na+-dependent bile acid transporter ASBT from Yersinia frederiksenii. Locations of putative (Acr3) or inferred from crystal structure (ASBT) transmembrane helices are highlighted in gray. Identical residues are in bold. Residues that are critical for the Na+/bile acid symport of ASBT or residues that are required for proper folding, sorting, or activity of Acr3p are in squares.
ACR3 RVLFEEVAKSVGVFLGIPLGIGIIIRLGSLTIAGKSNYEKYILRFISPWAMIGFHYTLFVIFISRGYQ 272 ASBT SVDVVGMLKSILQIVVIPITAGLVIHHT----------FTKTVKRIEPYLPAMSMVCILAIISAVVAG 207
ACR3 FIHE---IGSAILCFVPLVLYFFIAWFLTFALMRYLSISRSDTQRECSCDQELLLKRVWGRKSCEASF 337 ASBT SQSHIASVGFVVIIAVILHNGIGLLSGYWGGKLFGFDE------------------------------ 245
TM8 ACR3 SITMTQCFTMASNNFELSLAIAISLYGNNSKQAIAATFGPLLEVPILLILAIVARILKPYYIWNNRN 404 ASBT STCRTLAIEVGMQNSGLAATLGKIYFSP-----LAALPGALFSVWHNLSGSLLAGYWSGKPVKKDQE 307
and TM8–TM10) and a panel domain of four helices (TM1–TM2 and TM6–TM7). The most striking feature of the ASBT structure is that TM4 and TM9 are discontinuous helices (TM4a and TM4b; TM9a and TM9b) which cross each other. Two Na+-binding sites in ASBTYf, Na1 and Na2, are located behind the crossover region; Na1 involves Ser108 and Asn109 in TM4b, Glu254 in TM9a and Ser126 in TM5, while in the Na2 site, Na+ is coordinated by His71 in TM3 and Gln258 located between TM9a and TM9b (Fig. 7) (Zhou et al., 2014). Sequence alignment of ASBTYf with Acr3p revealed that both proteins possess similar transmembrane topology (Fig. 7). Remarkably, residues that are critical for the As(III)/H+ antiport are also located in TM4 (Cys151) and TM9 (Ser349, Phe352, Glu353). Thus, it is tempting to speculate that ASBT and Acr3 may be structural homologs and share a similar mechanism of transport. In support of this notion, crystallographic studies of two bacterial Na+/H+ antiporters, NhaA from Escherichia coli and NapA from Thermus thermophilus, showed the presence of the substrate-binding core domain with the crossover region composed of two antiparallel discontinuous helices (TM4a-b and TM11a-b), and the panel domain (Hunte et al., 2005; Lee et al., 2013). Two highly conserved aspartate residues, which are thought to function as an ion-binding site in TM5, are next to a crossover point in the core domain. It has been proposed that an ion binding to this site induces conformational changes in the adjacent crossover region due to an imbalanced electrostatic environment (Hunte et al., 2005; Lee et al., 2013). It is important to note that discontinuous helices implicated in transport mechanisms are commonly found in a wide range of secondary active transporters (Yamashita et al., © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 98, 162–174
2005; Faham et al., 2008; Weyand et al., 2008; Ressl et al., 2009; Shaffer et al., 2009). It has been suggested that both bile acid sodium symporters and Na+/H+ antiporters, like all secondary active transporters, seem to operate by an alternating access mechanism, in which the transporter exists in two major outward- and inward-facing states (Hunte et al., 2005; Hu et al., 2011; Lee et al., 2013; Zhou et al., 2014). The transition between outward and inward facing states involving partially or fully occluded substrate-bound transient states is also expected (Forrest et al., 2011; Kaback, 2015). It is thought that substrate binding to the core domain triggers conformation changes within the crossover region, causing rotation of the core domain against the panel domain; as a result of this, the substrate binding site moves for example from an inward to an outward position that enables the release of substrate on another site of the plasma membrane (Hunte et al., 2005; Hu et al., 2011; Lee et al., 2013; Zhou et al., 2014). Future crystallographic studies of the Acr3 family members are needed to establish whether As(III)/H+ antiporters operate by this mechanism.
Experimental procedures Strains, plasmids and growth conditions The acr3Δ::kanMX S. cerevisiae mutant (RW104) used in this study derives from the W303-1A wild-type (MATa ura3 leu2 trp1 his3 ade2 can1) (Wysocki et al., 2001). The pUG35 plasmid (CEN, URA3, AmpR) containing the ACR3-GFP fusion gene under the control of the native promoter (pACR3GFP) (Maciaszczyk-Dziubinska et al., 2010) was subjected to site-directed mutagenesis with the QuikChange Lightning
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kit (Agilent Technologies). Mutations in the ACR3 gene were confirmed by DNA sequencing. The acr3Δ::kanMX mutant was transformed with either the empty vector pUG35, pACR3-GFP or plasmids bearing the mutated versions of ACR3 gene. For As(III) sensitivity tests, the resulting transformants were grown to mid-log phase in selective minimal medium lacking uracil at 30°C, plated on solid selective minimal media containing various concentrations of sodium arsenite or potassium antimonyl tartrate and cultivated for 3 days at 30°C. For RNA isolation, total protein preparations and microscopy analysis, yeast transformants were grown to mid-log phase and exposed to 0.1 mM sodium arsenite for 4 h to induce expression of the ACR3 gene.
Measurements of As(III) efflux Exponentially growing cells were exposed to 0.1 mM As(III) for 2 h followed by 2 h incubation in the presence of 1 mM As(III). The As(III) content in cells was determined at the time of release from As(III) treatment and after 4 h of incubation in fresh medium using a flame atomic absorption spectrometer (3300, Perkin Elmer), as described elsewhere (MaciaszczykDziubinska et al., 2014).
Gene expression Total RNA was isolated from yeast transformants using RNeasy Mini Kit (Qiagen). Reverse transcription of ACR3 mRNA and real-time amplification of cDNA was performed as described previously (Maciaszczyk-Dziubinska et al., 2014).
Protein extraction and immunoblotting Total proteins were extracted with a trichloroacetic acid method and subjected to 10% SDS–PAGE followed by Western blot analysis. The anti-GFP antibody (Roche) at a dilution of 1:3000 was used to detect Acr3 tagged with a green fluorescent protein (GFP). Blots were also probed with the Cdc2 p34 (PSTAIRE) antibody (Santa Cruz Biotechnology) at a dilution of 1:3000 to examine the level of cyclindependent kinases Cdc28 and Pho85 as a protein loading control. Immunodetection of the membrane-enriched protein fraction was performed with the anti-Pma1 antibody at a dilution of 1:3000 against the plasma membrane H+-ATPase (kindly provided by M. Ghislain, University of Louvain, Belgium).
dextran/polyethylene glycol two-phase partitioning method as described previously (Norling, 2000; Maciaszczyk-Dziubinska et al., 2011; 2014). Protein content in plasma membrane vesicles was determined by the Bio-Rad protein assay reagent. The As(III)/H+ antiport activity was registered using acridine orange as a pH-sensitive probe. Membrane vesicles (50 μg) were added to 500 μL of assay buffer containing 10 mM of MES–Tris (pH 6.0), 330 mM of sucrose, 140 mM of KCl, 4 mM of MgCl2, 0.1 mM of ethylenediaminetetraacetic acid, 1 mM of dithiothreitol, 300 nM of bafilomycin A, 1 mM of NaN3, 0.1 mM of Na2MoO4, and 5 μM of acridine orange. Formation of the ΔpH gradient was initiated by addition of 2 mM of ATP, and the resulting change in acridine orange absorbance was monitored at 495 nm using a Beckman DU640 spectrophotometer. The ATP-dependent H+-transport activity was inhibited after 3 min by the addition of 0.5 mM of sodium orthovanadate. As(III)/H+ antiporter activity was measured based on its ability to dissipate the preformed, inside-acid pH gradient upon addition of 10 mM As(III). The acridine orange absorbance (A495) determined after proton gradient generation and immediately upon the addition of As(III) into the reaction medium containing the vesicles from different strains was set to the same value. The subsequent changes in A495 measured at various time points after addition of As(III) were calculated accordingly. The proton gradient generation is representative for plasma membrane vesicles isolated from different yeast strains. The As(III)-mediated dissipation of the proton gradient is representative for the results obtained separately for each strain with each measurement done in triplicate.
Sequence alignments and Acr3 topological model Genomic databases were screened with the S. cerevisiae Acr3 protein sequence using the BLAST program (Altschul et al., 1997). Alignment of protein sequences of representative members of the Acr3 family was performed with Clustal Omega (Sievers et al., 2011). A putative membrane topology model of S. cerevisiae Acr3p was predicted by the HMMTOP method (Tusnády and Simon, 2001).
Acknowledgements We greatly appreciate the gift of anti-Pma1 antibody from Dr. M. Ghislain (Université Catholique de Louvain, Louvain-laNeuve, Belgium). This work was supported by grant 2012/07/ B/NZ1/02804 from the National Science Centre, Poland.
Fluorescence microscopy The subcellular localization of Acr3-GFP proteins was determined in live cells with a fluorescence microscope (Axio Imager M2, Carl Zeiss) equipped with a 100× oil immersion objective, differential interference contrast and a GFP filter set. Images were collected using a Zeiss AxioCam MRm digital camera and processed with Zeiss Zen 2012 software.
Isolation of plasma membrane vesicles and As(III)/H+ antiporter assay The everted plasma membrane vesicles were prepared from yeast transformants treated with 0.1 mM of As(III) for 4 h by a
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