Genetic analysis of cytosolic iron toxicity in yeast

Iron is an essential nutrient for all eukaryotes and involved in many biological processes such as energy production, oxygen transport and DNA synthesis. Both iron deficiency and iron overload lead to human diseases. All organisms from yeast to humans have no regulated iron excretory pathway. Consequently, once iron enters cells it is detoxified by iron storage. In Saccharomyces cerevisiae, iron is stored in the vacuolar compartment mediated by the vacuolar iron transporter Cccl. Cells with a deletion of CCC1 are sensitive to high concentrations of iron. To understand the nature and origin of iron toxicity, we employed genetic screens to identify suppressors of high iron toxicity in \cccl cells. Our genetic analysis identified genes that reduced iron toxicity by decreasing cytosolic iron through increased iron sequestration in intracellular organelles. We identified that mutations in Zrcl, a vacuolar zinc and cobalt transporter, resulted in the ability to transport iron into the vacuole. We took advantage of these gain-of-function mutations to define amino acids and structural features important for substrate selection in the Zrcl family of cation diffusion facilitators. We also identified that overexpression of mitochondrial iron transporters Mrs3 or Mrs4 protected A cccl from high concentrations of iron by storing iron in mitochondria. Our genetic screen also identified Rim2 as a homologue of Mrs3 and Mrs4 and showed that Rim2 could also affect mitochondrial iron transport. Our studies identified novel forms of regulation of Mrs3/Mrs4 mediated iron transport. These genetic results suggest that iron induced damage occurs in the cytosol and iron sequestration in organelles can alleviate the toxic effect of high concentrations of iron.

GENETIC ANALYSIS OF CYTOSOLIC IRON TOXICITY IN YEAST by Huilan Lin A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Pathology Department of Pathology The University of Utah December 2009 Copyright © Huilan Lin 2009 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Huilan Lin This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. itti/ei rluAi Raymond Daynes / David Stillman John Phillips THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Huilan Lin jn jts form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. % ^ \ ^ Date Jerry\Kaplan"Y Supervisory Committee Approved for the Major Department Peter E. Jensen Chair/Dean Approved for the Graduate Council Charles A. Wight Dean of The Graduate School ABSTRACT Iron is an essential nutrient for all eukaryotes and involved in many biological processes such as energy production, oxygen transport and DNA synthesis. Both iron deficiency and iron overload lead to human diseases. All organisms from yeast to humans have no regulated iron excretory pathway. Consequently, once iron enters cells it is detoxified by iron storage. In Saccharomyces cerevisiae, iron is stored in the vacuolar compartment mediated by the vacuolar iron transporter Cccl. Cells with a deletion of CCC1 are sensitive to high concentrations of iron. To understand the nature and origin of iron toxicity, we employed genetic screens to identify suppressors of high iron toxicity in \cccl cells. Our genetic analysis identified genes that reduced iron toxicity by decreasing cytosolic iron through increased iron sequestration in intracellular organelles. We identified that mutations in Zrcl, a vacuolar zinc and cobalt transporter, resulted in the ability to transport iron into the vacuole. We took advantage of these gain-of-function mutations to define amino acids and structural features important for substrate selection in the Zrcl family of cation diffusion facilitators. We also identified that overexpression of mitochondrial iron transporters Mrs3 or Mrs4 protected A cccl from high concentrations of iron by storing iron in mitochondria. Our genetic screen also identified Rim2 as a homologue of Mrs3 and Mrs4 and showed that Rim2 could also affect mitochondrial iron transport. Our studies identified novel forms of regulation of Mrs3/Mrs4 mediated iron transport. These genetic results suggest that iron induced damage occurs in the cytosol and iron sequestration in organelles can alleviate the toxic effect of high concentrations of iron. v TABLE OF CONTENTS ABSTRACT iv LIST OF FIGURES viii LIST OF TABLES x ACKNOWLEDGEMENTS xi CHAPTER: 1. INTRODUCTION 1 Regulation of Systemic Iron Homeostasis 2 Diseases Due to Disrupted Iron Homeostasis 3 Regulation of Iron Homeostasis in Saccharomyces cerevisiae 6 The Role of the Yeast Vacuole in Metal Homeostasis 8 Regulation of Metal Homeostasis by Cation Diffusion Facilitators 9 Iron Transport into Mitochondria 11 Dissertation Preview 13 References 13 2. A SINGLE AMINO ACID CHANGE IN THE YEAST VACUOLAR METAL TRANSPORTERS ZRC1 AND COT1 ALTERS THEIR SUBSTRATE SPECIFICITY 22 Abstract 23 Introduction 23 Experimental Procedures 23 Results 24 Discussion 29 References 31 3. GAIN-OF-FUNCTION MUTATIONS IDENTIFY AMINO ACIDS WITHIN TRANSMEMBRANE DOMAINS OF THE YEAST VACUOLAR TRANSPORTER ZRC1 THAT DETERMINE METAL SPECIFICITY 32 Abstract 33 Introduction 33 Experimental 34 Results 35 Discussion 38 References 43 4. OVEREXPRSSION OF MRS3 AND MRS4 PROTECTS ACCC1 CELLS FROM HIGH IRON TOXICITY THROUGH ACCUMULATION OF IRON IN MITOCHONDRIA 44 Abstract 45 Introduction 45 Experimental Procedures 47 Results 50 Discussion 64 References 68 5. DISCUSSION 71 Determination of Substrate Specificity of Zrcl 72 Possible Mechanisms of Iron Toxicity 73 Conclusions 80 References 82 vii LIST OF FIGURES Figure Page 2.1 Acccl cells mutagenized by UV are resistant to high iron 25 2.2 Characteristics of high iron-resistant Acccl mutants 25 2.3 ZRC1(N441) confers resistance to high iron 26 2.4 ZRC1(N441) alters cytosolic iron by transporting iron into the vacuole 27 2.5 Iron transport efficiency of Zrcl(N44I) 28 2.6 Measurement of vacuolar iron content and Zrcl(N44I) protein abundance 28 2.7 ZRC1(N44I) shows an altered metal specificity 29 2.8 Alignment of Zrcl homologues 29 2.9 COTl(N45I) protects Acccl cells from high iron toxicity 30 2.10 COT1 is not involved in iron shock 30 3.1 Identification of transposon-generated genomic mutants that protect Acccl cells from high iron toxicity 35 3.2 Model for the topology of Zrcl 36 3.3 Zrcl mutants protect Acccl cells from high iron toxicity 38 3.4 Zrcl double mutants are not more efficient than single mutants in iron transport activity 39 3.5 Analysis of zinc transport activity in Zrcl mutants 39 3.6 All gain-of-function ZRC1 mutants confer resistance to high manganese toxicity 40 3.7 The effect of amino acid substitutions at position 40 and 44 of Zrc 1 on metal transport activity 40 3.8 Amino acid sequence alignment of S. cerevisiae Zrcl and E. coli YiiP 41 3.9 Model of the TMDs of Zrcl 42 4.1 Overexpression of Mrs3 and Mrs4 suppresses the toxic effect of high iron on Acccl cells 51 4.2 Mrs3 and Mrs4 are mitochondrial proteins 52 4.3 Overexpression of MRS3 increases mitochondrial iron 55 4.4 Overexpression of Mrs3 does not affect Sod2 activity 58 4.5 Overexpression of Mrs3 or Mrs4 does not suppress Acccl when grown anaerobically 60 4.6 Mrs3 and Mrs4 expression levels and localization are not changed when cells are grown anaerobically 62 4.7 Mitochondrial mass does not change when cells are grown anaerobically 65 5.1 Magnesium supplementation protects Acccl cells from high concentrations of iron 79 5.2 Alcohol dehydrogenase activity is not affected in Acccl cells 81 ix LIST OF TABLES Table Page 3.1 Primers used in the present study 34 3.2 Nucleotide and amino acid changes in mutant Zrcl 37 3.3 Summary of the Zrcl mutants in the present study 41 ACKNOWLEDGEMENTS This dissertation would not be possible without the help of many mentors. First of all, I would like to thank my advisor Dr. Jerry Kaplan for his continuous support. Jerry has been an excellent mentor and always provided unique insight to move my research forward. I would also like to thank Dr. Diane McVey Ward in the sharing of scientific ideas, techniques and writing of manuscripts. I would also like to thank all members of the Kaplan/Ward lab for sharing their ideas and knowledge. Especially Opal Chen for guiding me through yeast genetics, Attila Kumanovics and Liangtao Li for technical assistance, and Dave Warner and Jenifer Nelson for their participation in these projects. My thesis committee members, Drs. Raymond Daynes, David Stillman, Dennis Winge and John Phillips, gave valuable assistance in directing the research projects, providing reagents, methods and allowing me use of equipment. Their efforts are greatly appreciated. My wife, my parents and my brother and sisters have given me enormous support and encouragement throughout these years. A special thank is due to them. I would also like to thank Portland Press Ltd. for permission to use the following article: Lin H, Burton D, Li L, Warner DE, Phillips JD, Ward DM, Kaplan J. Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrcl that determine metal specificity. Biochem J. 2009 Aug 13; 422(2):273-83. Copyright © 2009 Biochemical Society. CHAPTER 1 INTRODUCTION 2 Regulation of Systemic Iron Homeostasis Iron containing metalloproteins (hemeproteins, iron-sulfur proteins, and other iron-dependent enzymes) are involved in many biochemical processes, such oxygen transport, electron transfer and DNA synthesis (1). This makes iron indispensable for life. Iron can exist as two different oxidation states, ferrous and ferric. This redox property makes iron ideal for carrying out oxidation-reduction reactions when appropriately coordinated. However, these same redox properties make iron a potent catalyst for reactions with oxygen making iron potentially toxic to organisms. Iron can react with molecular oxygen to generate superoxide anions, hydrogen peroxide and hydroxyl radicals. These reactive oxygen species can damage proteins, lipids and DNA molecules. Due to the essential and toxic character of iron in all species the level of iron in all biological fluids is tightly controlled. Since there is no regulated excretion pathway for iron once iron enters the body, iron absorption is tightly regulated to meet the iron demands of organisms (2, 3). In vertebrates, iron is transported into the duodenum through the divalent metal transporter 1 (DMT1) at the apical surface of absorptive enterocytes (4, 5). Iron (ferrous) absorbed by the enterocytes is then exported to the serum through the iron exporter ferroportin-1 (FPN1) (6) at the basolateral surface, where ferrous iron is oxidized to ferric iron by hephaestin (Hp), a ferroxidase (7). The ferric iron is then bound to transferrin with high affinity and goes into the circulation. The antimicrobial peptide hepcidin is a key regulator of systemic iron homeostasis. Hepcidin is produced in liver (8) in response to inflammatory cytokines such as IL-6 (9) and iron overload (10, 11). Hepcidin binds to its plasma membrane receptor ferroportin and induces its degradation (12), leading to less iron entering the plasma through the basolateral surface. Thus hepcidin is a negative regulator of intestinal iron absorption. When iron in the circulation is high, hepcidin is induced to downregulate ferroportin, limiting iron entrance to the plasma. When iron in the plasma is low, hepcidin expression is low and ferroportin is stabilized permitting export of iron into the plasma. Diseases Due to Disrupted Iron Homeostasis Iron Deficiency Diseases Iron is absorbed from the duodenum at 1-2 mg/day (2). If there is insufficient iron in the diet, iron deficiency will lead to anemia. This nutritional iron deficiency anemia affects 1 to 2 billion people worldwide (13). Iron is important for the development of the brain and iron deficiency can lead to morphological changes in the hippocampus and striatum (14, 15). Iron deficiency can also alter neurometabolism such as monoamine metabolism (16). As a result, human infants show reduced cognitive and learning capability. This kind of iron deficiency can be prevented by dietary iron supplementation. Iron deficiency can also result from genetic alterations (17). The microcytic anemia (mk) mouse has impaired DMT1 function (4). Sex-linked anemia (sla) in mice is due to the loss of hephaestin (7). The hypochromic anemia of the zebrafish mutant Weissherbst is due to loss of ferroportin activity (18). These genetic mutants have helped to delineate the iron uptake pathway in the intestine, where the mRNA levels of DMT 1, hephaestin and ferroportin are all increased to compensate for the body iron demand during iron deficiency (19). 4 Iron Overload Diseases The tight regulation of iron homeostasis ensures that iron absorption meets the iron demand to limit excess iron accumulation in the body. Some genetic defects leading to increased iron absorption or transfusional iron loading during treatment of anemia, can result in iron deposition in liver and other parenchymal tissues leading to liver cirrhosis, diabetes and cardiac disease (20). Hemochromatosis is the most common iron overload disease. Common biochemical manifestation includes elevated serum transferrin saturation and ferritin levels. This is largely due to increased iron release from enterocytes and macrophages into the blood stream. There are four types of hereditary hemochromatosis (21). Type 1 is due to mutations in HFE; Type 2 can be divided into two subtypes, type 2A is due to mutations in hepcidin and type 2B is due to hemojuvelin (HJV) mutations; type 3 is caused by a transferrin receptor 2 (TfR.2) mutation; and type 4 is due to ferroportin mutations. The first three forms are recessive diseases while type 4 shows dominant characteristics. The interaction between hepcidin and ferroportin provides the underlying mechanism for this iron overload disease. HFE, TfR2 and hemojuvelin are all involved in regulation of hepcidin. Mice homozygous for deletion of HJV (22), deletion of HFE (23) or with a TfR2 mutation (24) all show decreased expression of hepcidin. The defect in hepcidin production leads to uncontrolled iron release from intestinal cells and macrophages resulting in iron accumulation in the plasma and ultimately in parenchymal tissues. Mutations in ferroportin fall into two classes; proteins that are unable to target to the plasma membrane and proteins that are unable to respond to hepcidin-induced degradation. Depending on the nature of the Fpn mutations, the resulting phenotype can 5 be Kiipffer cells iron overload (mutants unable to localize to the cell surface) or hepatocytes iron overload (mutants unable to respond to hepcidin) (25). Congenital hypotransferrinemia or atransferrinemia is another form of iron overload disease found in humans and mice (26, 27). Transferrin binds ferric iron and delivers iron into erythrocytes for hematopoiesis. In the absence of sufficient transferrin, mice and humans are anemic. This anemia increases iron absorption from the gut (28), and non-transferrin-bound iron is delivered and deposited in soft tissues (29, 30) leading to iron overload. Aceruloplasminemia is an autosomal recessive disease due to mutations in the gene encoding ceruloplasmin, leading to the absence of circulating serum ceruloplasmin (31). Ceruloplasmin is a copper containing ferroxidase and catalyzes the oxidization of ferrous iron to ferric iron to load transferrin (32). Ceruloplasmin mediates iron efflux from reticuloenduothelial cells, hepatocytes (33, 34) and astrocytes of the central nervous system (34). It affects iron efflux probably through stabilizing ferroportin expression on the cell surface (35). Aceruloplasminemia patients accumulate iron in the brain and show symptoms of neurodegeneration (32). Friedreich ataxia (FRDA) is an iron overload disease in mitochondria. Friedreich ataxia an autosomal recessive degenerative disease affecting about 1 in 40,000 individuals (36). The patients show progressive limb and gait ataxia as well as dysarthria, areflexia, sensory loss and muscle weakness (36). The genetic basis is a GAA trinucleotide expansion in the first intron of the frataxin gene (37), which leads to decreased expression of frataxin mRNA and protein in the mitochondria (38). Frataxin is a mitochondrial protein involved in iron-sulfur cluster synthesis (39-41) and heme synthesis (42, 43). Patients with FRDA show aeonitase and mitochondrial iron-sulfur protein deficiency (44). Yeast cells lacking the frataxin homolog (Yfhl) and patients with FRDA accumulate iron in the mitochondria (45, 46). Mitochondrial iron accumulation, however, may be a secondary effect of loss of frataxin, as depletion of other components of the mitochondrial iron-sulfur synthesis machinery also lead to iron accumulation in mitochondria (47). Iron accumulation in mitochondria may generate reactive oxygen species that lead to oxidative damage to proteins (48), DNA (49, 50) and lipids (50). Treatment for patients with FRDA has been focused on decreasing the production of free radicals and preventing iron accumulation using iron chelators (36). Regulation of Iron Homeostasis in Saccharomyces cerevisiae Many of the proteins involved in iron transport and metabolism are conserved from yeast to human. Saccharomyces cerevisiae has been used as a model organism to study iron metabolism. Yeast, like humans, has no excretory pathway and control iron homeostasis by regulating iron absorption and iron storage. This homeostasis is achieved through transcription factors that tightly coordinate the expression of genes involved in iron uptake, utilization, and storage (51, 52). Response to Iron Deprivation Two transcription factors Aftl (53) and Aft2 (54) play an important role in response to iron deprivation. Aftl is constitutively expressed and is located in the cytosol when iron is abundant. When iron is depleted, Aftl moves into the nucleus (55) and activates the set of genes collectively called the iron regulon (56, 57). These gene products include: 1) cells wall mannoproteins Fitl-3p that facilitate the retention of siderophores; 2) metalloreductases Frel-4 that reduce Fe(III) to Fe(ll), the high affinity iron uptake complex composed of Fet3/Ftrl, and the siderophore transporters Arnl-4; 3) proteins that mobilize iron stored in vacuoles into cytosol such as the metalloreductase Fre6, the iron transporters Smf3 and Fet5/Fthl; 4) heme degradation protein Hmxl (58); 5) RNA binding proteins Cthl/2 that assist in the degradation of mRNAs encoding proteins involved in iron storage and iron utilization such as Cccl, Acol, Sdh4 (59, 60). Aft2 is a paralogue of Aftl and can activate the iron regulon independent of Aftl. Aft2 directly activate Smfi and Mrs4 (57), transporters involved in vacuolar and mitochondrial iron homeostasis respectively. It is suggested that Aft2 is mainly involved in regulating organelle iron homeostasis. Response to Iron Excess When sufficient iron is supplied to iron-depleted yeast, the transcription factor Aftl translocates to the cytosol (55) and its target genes are turned off. The high affinity iron uptake complex Fet3/Ftrl is internalized and degraded in the vacuole. Ubiquitination is involved in the degradation of the Ftrl/Fet3 complex. Ubiquitination is mediated by the E3 ubiquitin ligase Rsp5 and occurs in the endosome (61, 62). The transcription of CCC1, which encodes the vacuolar iron transporter, is increased through the action of the transcription factor Yap5. Yap5 binds constitutively to the TTACTAA sequence in the promoter region of CCC1. In response to high cytosolic iron, the disulfide bonds of Yap5 DNA activation domain is modified and this modification regulates Yap5 transcriptional activity (63). Whether this modification is a direct effect of iron or involves other modifying proteins is not clear. Cells with a deletion of YAP5 or CCC1 are unable to grow on medium containing high concentrations of iron. Other proteins involved in the cell adaptation to high concentration of iron include Csg2, Pho80, Rad57 and Vam7, as deletion of the genes that encode these proteins results in reduced growth in medium containing high concentrations of iron (64). The Role of the Yeast Vacuole in Metal Homeostasis The yeast vacuole plays an important role in metal homeostasis. Vertebrates store iron in cytosolic ferritin but plants and fungi store excess iron in the vacuole, which is functionally similar to lysosomes. Vacuoles/lysosomes are acidic organelles that contain high concentrations of hydrolases that are capable of degrading most biological macromolecules. Yeast cells that lack vacuoles, or are unable to acidify their vacuoles due to mutations in the V-ATPase (65, 66), accumulate less metal in the cytosol and show sensitivity to high concentrations of metals. Studies show that many divalent cations such as Fe, Co, Mn, Ni, Zn, Cd, and Cu are stored and detoxified in vacuoles. Specific vacuolar transporters mediate the transport of metals from cytosol to vacuole. An ATP binding cassette (ABC) transporter Ycfl transports cadmium glutathione conjugates (Cd*GS2) into vacuoles and contributes to cadmium detoxification (67, 68). Yeast can also accumulate nickel in the vacuole, although the transporter for nickel has not been identified (69). Zrcl and Cotl are two homologous proteins belonging to the cation diffusion facilitator family, which transport zinc and cobalt into the vacuole. These proteins are H+/M21 antiporters and utilize the high H+ concentration of the vacuole to transport cytosolic metals into the vacuole. Cells with deletions of ZRC1 or COT1 are sensitive to zinc (70), while cells that overexpress ZRC1 or COT1 are more resistant to zinc and cobalt respectively (71, 72). Cccl transports Fe into the vacuole (66), but Cccl does not belong to the CDF family. Cccl and its plant homologues VIT1 are a small unique family, \cccl cells are sensitive to high concentrations of iron while cells overexpressing CCC1 accumulate more iron in the vacuole. Genetic studies suggest that Cccl is also involved in manganese and calcium transport into the vacuoles (73, 74). Overexpression of CCC1 can suppress the manganese sensitivity of Apmrl cells. Pmrl is a Golgi membrane P-type ATPase transporter that transports manganese and calcium into the secretory pathway (75). Overexpression of CCC1 is able to rescue the calcium sensitive phenotype of csgl mutants (73). When cells are shifted from metal abundant medium to metal deficient medium, metals stored in the vacuole can be mobilized by specific transporters and utilized for metabolic purposes. Iron in the vacuole is reduced by the metalloreductase Fre6 and transported into the cytosol by Smf3 and Fet5/Fthl (76). Zrt3 mobilizes zinc stored in the vacuole when cells are limited for zinc. Transcription of ZRT3 is upregulated by the Zapl transcriptional activator (77). Copper stored in the vacuoles can also be mobilized into the cytosol. The ferrireductase Fre6 and the vacuolar copper exporter Ctr2 are involved in this process (78, 79). The vacuolar copper importer has not been identified yet. Regulation of Metal Homeostasis by Cation Diffusion Facilitators Cation diffusion facilitator (CDF) transporters are a large family involved in metal homeostasis. They are found in all three kingdoms of life. They show great diversity in cellular localization, substrate specificity and tissue specific expression. They are involved in transporting Fe, Zn, Co, Cd, Mn, and Ni out of the cell or into intracellular organelles, thus reducing cytosolic metal concentration. E. coli ZitB (80) and mammalian ZnT-l(Slc30al) (81), which mediate zinc efflux from cells, are expressed on the plasma membrane. S. cerevisiae Zrcl and Cotl are vacuolar proteins that transport zinc and cobalt into vacuoles for storage and detoxification (71, 72, 82, 83). Arabidopsis thaliana MTP11 localizes to pre-vacuolar compartments and mediates manganese tolerance (82, 83). ZnT-8 is specifically expressed in pancreatic beta cells and transports Zn into insulin containing vesicles for insulin maturation (84). In S. cerevisiae, two CDF transporters Msc2 and Zrgl7 form a heteromeric complex in the ER membrane and transport Zn into the ER. Their mammalian homologues ZnT5 and ZnT6 also interact with each other and transport zinc into the secretory pathway of mammalian cells (85). Despite differences in location and metal specificity, CDF members share some common features. The majority of CDF family members have six putative transmembrane domains (TMD) with the amino and carboxyl termini extending into the cytosol. A highly conserved amino acid sequence extending from TMD II to III, is a signature motif for the family (86). Based on the alignment of multiple sequences, highly conserved charged residues in TMD II and V are implicated in metal binding and transport. This finding is supported by structural studies on an E.coli CDF family member FieF (also known as YiiP), a putative Zn2+ and Fe2+ transporter that mediates iron resistant growth of E. coli. YiiP is a homodimer (87). In the transmembrane domain, each monomer contains one zinc-binding site formed by four charged residues in TMD II and V. In the carboxyl terminal cytosolic domain, each monomer contains three-zinc binding sites (88). The eytosolic domain may be involved in sensing the cytosolic zinc concentration. Structural study of the cytosolic domain of CzrB, a CDF family zinc transporter from Thermus thermophilics, shows that the cytosolic domain forms a dimer and changes its conformation upon zinc binding. The conformational change may bring the transmembrane domains of two monomers together to facilitate zinc transport (89). Based on the conformational change, a metallochaperon is proposed to deliver zinc to the transporter. The details of transport are still unclear, as there is no metal channel formed by each individual transmembrane domain. Iron Transport into Mitochondria Mitochondria house the biosynthetic enzymes for the synthesis of two important cofactors involved in iron metabolism: iron sulfur clusters (47, 90) and heme (91). Iron must cross the mitochondria inner membrane to serve as a substrate for iron sulfur scaffold proteins or for ferrochelatase, the enzyme that inserts iron into the protoporphyrin ring to make heme. The mitochondrial carrier family members Mrs3 and Mrs4 are implicated in transporting iron into mitochondrial. Deletion of both MRS3 and MRS4 suppresses the iron accumulation phenotype of yeast cells with a deletion of the yeast frataxin homolog YFH1 (92). Single deletions of MRS3 or MRS4 have no phenotype while a double deletion of MRS3 and MRS4 shows a growth defect in low iron medium. Consistent with this phenotype, heme synthesis and iron incorporation into iron sulfur cluster containing proteins are impaired only under iron-limited conditions. When cells are grown in iron-replete medium, there is no difference between wild type strain and the double deletion strain (93). These results suggest that Mrs3/Mrs4 are high affinity 12 iron transporters. When cytosolic iron is high, mitochondrial low affinity iron transporters are sufficient to provide iron to mitochondria in the absence of Mrs3 and Mrs4. The identity of these low affinity iron transporters is still unknown. A more direct measure of iron transport into mitochondria using submitochondrial particles and the quenching of the iron sensitive fluorophore PhenGreen SK shows that Smrs3Smrs4 mitochondria take up iron slower than wild type mitochondria while mitochondria overexpressing MRS3 and MRS4 take up iron more rapid than wild type (94). Mitoferrinl and mitoferrin2 from zebrafish and mammals are homologues of yeast Mrs3 and Mrs4 (95). Defects in mitoferrinl lead to erythroid maturation arrest in zebrafish (95). Iron incorporation into heme is dramatically reduced in murine embryonic stem cells null for mitoferrin. The low iron growth defect of yeast Es.mrs3S.mrs4 cells can be rescued by expressing zebrafish mitoferrinl or mitoferrin2 (95). Mammalian cells silenced for mitoferrinl and mitoferrin2 show decreased mitochondria iron, heme and iron sulfur cluster synthesis (96). These data indicate that Mrs3/4 and their mammalian homologues have a conserved function in transporting iron into mitochondria. Two proteins Mmtl and Mmt2 have also been implicated in mitochondrial iron transport. Overexpression of MMT1 and MMT2 is able to suppress the low iron growth defect of erg25-2 cells (97). Erg25 is an iron-containing enzyme involve in ergosterol synthesis. The mutant enzyme, Erg25-2 is thought to lose iron when cytosolic iron levels are low (97). When overexpressed, Mmtl or Mmt2 increases mitochondrial iron content (98). Deletion of both MMT1 and MMT2 shows no obvious iron related phenotype. The existence of other iron transporters on mitochondria is implied by the fact that a quadruple mutant ts.mrs3Smrs4SmmtltS.mmt2 is still viable. Dissertation Preview Iron overload disease in humans leads to substantial pathology. To understand the mechanisms responsible for the toxicity of high concentrations of iron, we employed a genetic approach using Saccharomyces cerevisiae. We took advantage of the finding that deletion of CCC1, the gene that encodes the vacuolar iron transporter, results in cell death under high concentration of iron conditions. We employed genetic approaches to isolate mutants that were able to suppress high iron sensitivity of \cccl cells. Analysis of iron-resistant mutants led to the following findings: 1) Amino acid substitutions in the CDF family member zinc transporter Zrcl permitted this transporter to lower cytosolic iron by transporting iron into vacuoles. We took advantage of the finding that mutations in Zrcl could result in a gain-of-function to analyze amino acids required for substrate specificity. 2) Cytosolic iron toxicity could be reduced by storage of iron in mitochondria through overexpression of mitochondrial iron transporters Mrs3 or Mrs4. References 1. 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Printed in the U.S.A. A Single Amino Acid Change in the Yeast Vacuolar Metal Transporters Zrcl and Cotl Alters Their Substrate Specificity* Received for publication, June 6,2008, and in revised form, October 10,2008 Published, JBC Papers in Press, October 16,2008, DOI 10.1074/jbc.M804377200 Huilan Lin, Attila Kumanovics1, Jenifer M. Nelson, David E.Warner, Diane McVey Ward, and Jerry Kaplan2 From the Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132 Iron is an essential nutrient but in excess may damage cells by generating reactive oxygen species due to Fenton reaction or by substituting for other transition metals in essential proteins. The budding yeast Saccharomyces cerevisiae detoxifies cytosolic iron by storage in the vacuole. Deletion of CCC1, which encodes the vacu-olar iron importer, results in high iron sensitivity clue to increased cytosolic iron. We selected mutants that permitted A cccl cells to grow under high iron conditions by UV mutagenesis. We identified a mutation (N44I) in the vacuolar zinc transporter ZRC1 that changed the substrate specificity of the transporter from zinc to iron. COT1, a vacuolar zinc and cobalt transporter, is a homologue of ZRC1 and both are members of the cation diffusion facilitator family. Mutation of the homologous amino acid (N45I) in COT1 results in an increased ability to transport iron and decreased abil-ity to transport cobalt. These mutations are within the second hydrophobic domain of the transporters and show the essential nature of this domain in the specificity of metal transport. The yeast vacuole plays an important role in transition metal storage and detoxification. In conditions of metal scarcity, met-als stored in the vacuole can be mobilized by specific transport-ers and utilized for metabolic purposes. Conversely, export of metals from cytosol to vacuole is thought to prevent metal tox-icity. Yeast mutants that are unable to store iron in the vacuole, either due to a lack of vacuolar structures (1), an inability to acidify vacuoles due to mutation in the V-ATPase (2, 3), or deletion of vacuole metal transporters (4 - 6) show sensitivity to high concentrations of metals. Specific transporters mediate the transport of different tran-sition metals from cytosol to vacuole. Among the best studied of the vacuolar transition metal transporters are the zinc trans-porters Zrcl and Cotl (4, 5, 7-9). These homologous proteins, which are involved in the transport of zinc and cobalt are mem-bers of the cation diffusion facilitator (CDF) 5 family (for review * This work was supported, In whole or in part, by National Institutes of Health Grant DK30534 (to J. K.), support for use of the Core Facilities was provided through NCI, National Institutes of Health Grant NCI-CCSG P30CA 42014 and NIDDK Center of Excellence Award 5P30KD72437. The costs of publi-cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement' in accord-ance w i t h 18 U.S.C. Section 1734 solely to indicate this fact. ' Partially supported by National Institutes of Health Training Grant T32 DK07115-29. 2 To w h o m correspondence should be addressed. Tel.: [phone]; Fax: [phone]; E-mail: [e-mail]. 3 The abbreviations used are: CDF, cation diffusion facilitator family; BPS, bathophenanthroline disulfonate; CM, complete medium; ICP, inductively see Ref. 10). Although CDF members show differences in size, cellular localization, and substrate metals transported, they share some common features. The majority of CDF family members have six putative transmembrane domains (TMD) and a highly conserved amino acid sequence extending from TMD II to III, which is a signature motif for the family. Based on the alignment of multiple sequences, highly conserved charged residues in TMD II and V are implicated in metal binding and transport. This finding is supported by structural studies on an Escherichia coli CDF family member Fief (also known as YiiP), a putative Zn2 + and Fe2+ transporter (11, 12). Transport of iron from cytosol to vacuole is mediated in yeast by Cccl (6) and in plants by the CCC1 homologue VIT1 (13). These proteins define a unique family that appears to be restricted to fungi and plants. Little is known of the mechanism of transport, although it is clear that CCC1 is regulated by iron at transcriptional and post-transcriptional levels (14,15). Dele-tion of CCC1 results in poor growth in high iron medium indi-cating that increased cytosolic iron may be toxic (6). The mech-anising) leading to high iron toxicity is unknown. High iron toxicity occurs in cells deleted for CCC1 even in the absence of respiratory capacity (e.g. rho° cells) or anaerobically (16). These results call into question the assumption that high iron toxicity is due to the generation of iron-mediated reactive oxygen rad-icals. In an effort to define the mechanism of iron toxicity we initiated a genetic screen in which we identified mutant strains of A cccl that grew on high iron. Herein, we identify a missense mutation in the vacuolar zinc transporter Zrcl, which com-pletely changes the substrate specificity of this transporter from Zn2 ' to Fe2+. We show that a similar amino acid change in the homologous Cotl, a Co2 + and Zn2 + transporter, also results in a change in metal specificity. EXPERIMENTAL PROCEDURES Yeast Strains-The following yeast strains (W303 back-ground) were used: DY150 (Mata ade2-l his3-U leu2-3,112 trpl-1 ura3-S2 canl-lOO(oc)) and DY1457 (Mataade6 his3-U leu2-3,112 trpl-1 ura3-S2 canl-100(oc)). Deletions of CCC1 and ZRC1 were generated by double fusion polymerase chain reaction using the H1S3 gene as a selectable marker (17). Prim-ers for disruption of CCC1 were described (6). Primers for dis-ruption ofZRC1 were Pri20-78 (5'-TCT CTT TTG ACC TTA GAC ACG-3'), Pri20-79 (5'-GTC GTG ACT GGG AAA ACC coupled plasma-optical emission spectrometer; TMD, transmembrane domain; YPD, yeast extract peptone dextrose medium. DECEMBER 5, 2008-VQLUME 283-NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33865 24 Metal Specificity of Vacuolar Transporters CTG GCG ATC GCT GCC ATG ATC GTG GAA-3'), Pri20-80 (TCC TGT GTG AAA TTG TTA TCC GCT GCT GAT CAG ATT CAA AGA GAG-3'). and Pri20-81 (GCA GTT TAC AGC GTC ATC TAC-3'). The C0T1 gene was dis-rupted using URA3 as described (4). Strains with a FET3-lacZ reporter integrated at the HO locus were constructed as described (18). Wild type BY4743 (Mat ala his3Allhis3Al leu2A0lleu2A0 lys2A0l+ met 15 AO/+ ura3AOIura3AO), Acotl\:KanMX and Apmrlv.KanMX strains in the BY4743 background were obtained from Research Genetics. Growth Media-Yeast strains were'grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or in CM medium (0.67% yeast nitrogen base without amino acids, 2% dextrose, and 0.13% amino acid drop-out mixture). Low iron growth medium was made by adding 40 fj.M bathophenanthroline dis-ulfonate (BPS), an iron chelator, to CM and then the addition of specified amounts of FeS04. To make high iron plates, ferrous ammonium sulfate (250 mM stock in water) was added into media to give the indicated iron concentrations. FeS04 (50 mM stock in 0.1 M HC1) was used in liquid culture as indicated together with a final concentration of 1 mM ascorbic acid. Isolation of Mutants-DY150 Acccl or DY1457 Acccl cells were plated on CM agar plates at a density of 500 cells/plate and exposed to UV light to give a 50% survival. Mutagenized cells were grown for 2 days and then replicated to CM plates con-taining 5 mM ferrous ammonium sulfate. Cells were grown for another 3 days. Colonies able to grow on high iron were selected for further study. Identification of ZRC1(N44I)-The mutated gene in domi-nant mutant R1 was identified by constructing a library from the R1 genomic DNA using standard protocols (19). Briefly, Sau3A partially digested genomic DNA was fractionated on a 10-40% sucrose gradient, and centrifuged at 25,000 X g for 22 h. The 8-10-kb genomic fraction was ligated to pRS416- zero, a centromeric plasmid, digested with BamHI and dephos-phorylated with calf intestinal phosphatase (New England Bio- Labs). Ligation products were transformed into Electromax1M DH10B™ E.coli (Invitrogen) (20). Plasmids were extracted from transformed bacteria and pooled. The pooled library was transformed into Acccl cells and plasmids that conferred resist-ance to high iron (3 mM) were recovered. The genomic frag-ments in these plasmids were identified by sequencing. Plasmids Construction and Site-directed Mutagenesis- ZRC1(N44I) was subcloned from the genomic library using Kpnl and Bglll and inserted into pRS416 (a yeast centromeric vector), which had been digested with BamHI and Kpnl. Wild type ZRC1 with its own promoter and 3' end was generated using PCR from genomic DNA using pri54 (5'-GAT ATG AAA GTA GTT GCA TT-3') and pri60 (5'-TTG GTA CAG GAG GGA ACA AG-3'). The PCR fragment was digested with Bglll and Kpnl and inserted into pRS416 digested with BamHI and Kpnl. To introduce the N44I mutation into wild type ZRC1, pri64 (5'-GGC CTT GAT TGC CGA TTC ATT TCA CAT GTT GAT TGA TAT CAT CTC TCT TTT AGT GGC-3') and pri65 (5'-GCC ACT AAA AGA GAG ATG ATA TCA ATC AAC ATG TGA AAT GAA TCG GCA ATC AAG GCC-3') were used. To introduce the D45A mutation into wild type ZRC1, pril02 (5'-CAC ATG TTG AAT GCT ATC ATC TCT CTT TTA GTG GCA C-3') and pril03 (5'-GTG CCA CTA AAA GAG AGA TGA TAG CAT TCA ACA TGT G-3') were used. COT1IYEp352 was obtained from Dr. Douglas S. Conklin (4). To introduce N451 into wild type COT1, pri66 (5'-CGC GGA CTC ATT CCA TAT GCT AAT CGA TAT AAT TTC TCT TGT GG-3') and pri67 (5'-CCA CAA GAG AAA TTA TAT CGA TTA GCA TAT GGA ATG AGT CCG CG-3') were used. Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit from Stratagene according to the manufacturer's instructions. ZRC1 and ZRC1(N44I) were digested with Kpnl and Xbal and inserted into a yeast episomal vector pTF62 (LEU2 marker). To generate His6-tagged versions of CCC1, ZRC1, and ZRC1(N44I) under the control of the galactose inducible promoter (GAL1) the fol-lowing primers were used: CCC1, pri78 (5'-CGC GGA TCC ATG TCC ATT GTA GCA CTA AAG-3') and pri79 (5'-CCG GAA TTC ACC CAG TAA CTT AAC AAA GAA-3'), for ZRC1 and ZRC1(N44I), pri80 (5'-GGG CGA AGA TCT ATG ATC ACC GGT AAA GAA TTG-3') and pri81 (5'-CCG GAA TTC CAG GCA ATT GGA AGT ATT GCA-3'). DNA was amplified by PCR to generate each open reading frame without the stop codon. The amplified products were inserted into BamHI- and EcoRI-digested pYES2/CT (Invitrogen). Metal Analysis-For whole cell metal analysis, 20 OD cells (about 2 X 10s cells) at log phase were collected, washed three times with 50 mM Tris-Cl (pH 6.5), 10 mM EDTA and once with deionized water. Vacuoles were prepared using Ficoll gradients as described previously (6). Samples were digested in 200 /xl of nitric acid at 80 °C for 1 h, then diluted to 1.0 ml with deionized water. Metals were analyzed in a Perkin-Elmer Inductively Coupled Plasma-Optical Emission Spectrometer (ICP) and cal-culated using a standard curve generated from mixed metal standards. Western Blot, ji-Galactosidase Activity, and Protein Concen-tration Assay-Cells were disrupted with glass beads in the presence of protease inhibitors (1.0 mM phenylmethylsulfonyl fluoride, 1.0 /xm pepstatin A, and 1.0 /am leupeptin) (Sigma). Samples (20 jj,g) were run on a 12% SDS-PAGE, transferred to nitrocellulose, and probed with rabbit anti-His6 tag (1:2000 Abeam) or mouse anti-Vmal antibody (1:4000, Molecular Probes), followed by peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody (1:10,000, Jackson Immuno- Research). Membranes were developed using chemilumines-cence reagents (PerkinElmer Life Sciences). The reporter con-structs FET3-lacZ and CCCl-lacZ were described previously (15, 18). j3-Galactosidase activity was performed in 96-well plates using ort/!o-nitrophenyl-)3-galactoside as a substrate. The generation of orrto-nitrophenol was monitored at 420 nm and the data are presented as nanomole/min/mg of protein (18). Protein concentration was determined by the bicincho-ninic acid method (Pierce) using bovine serum albumin as standard. RESULTS Identification of Yeast Mutants Resistant to High Iron-Dele-tion of CCC1 results in sensitivity to high iron (6, 15). We took advantage of this phenotype to identify UV-induced mutants of Acccl cells that were capable of growth on high iron medium. 33866 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 49 - DECEMBER 5, 2008 25 Metal Specificity of Vacuolar Transporters A o.s CM 3 mM Fe DYI50 DYI457 Acccl W17 n m l X X B © a ® * © W i® If * - • • a ? • • • <> • in DY150 Acccl Rl DYI457 Acccl W2 VV7 \VI7 2(iMFe III ^M Fe 1 r i \ 0.005% H202 2.5 mM paraquat FIGURE 1. Acccl cells mutagenized by UV are resistant to high iron. A, DY150 (wild type), Acccl, and the Acccl mutants isolated after UV mutagenesis. Rl, W2, W7, and W17 were g r o w n to log phase in CM medium and then inoculated at a starting OD of 0.005 i n t o CM medium w i t h ferrous ammonium sulfate at the indicated concentrations. Cells were incubated at 30 °C overnight and the cell density was measured by absorbance at 600 nm. The experiment was performed multiple times and a representative experi-ment is presented. B, DY150 (wild type), DY150 Acccl, Rl mutant, DY1457 (wild type), DY1457 Acccl, and W2, W7, and W17 mutants were grown to log phase in CM medium and 10'', 1 0 3 , 1 0 2 , a n d 10' cells spotted onto CM plates or CM plates with 3 mM ferrous ammonium sulfate. Plates were incubated at 30 °C for 2 days. The color difference of the colonies is due to different ade mutations (see "Experimental Procedures" for genotype). We screened 12,400 colonies and identified 15 mutants that would survive on high iron medium. The mutants were back-crossed to the parental strain (Acccl) to determine whether they were single gene defects and whether the mutants were dominant or recessive. Seven of the mutants were single gene recessive mutations that fell into two complementation groups. Eight of the mutants were dominant. The mutants were char-acterized as having different sensitivity to high iron, as shown by liquid (Fig. \A) and plate growth assays (Fig. IB). There are several potential mechanisms that might lead to high iron-resistant growth: decreased iron uptake, increased iron export, increased iron storage, or increased antioxidant defenses. To determine whether the mutant strains had decreased iron acquisition, cells were grown in CM medium overnight and cellular iron levels were measured by ICP. Mutants W2 and W7 accumulate less iron than control A cccl cells. Rl and W17 accumulated more iron than the parental A cccl strain (Fig. 2A). If resistance to high iron was due to increased iron export from cells, then the mutant strains might not grow on low iron. This situation was observed for a mutant PDR1 in which high iron resistance was correlated with low iron sensitivity (21). The A cccl mutant strains were able to I2Y1457 A cccl W2 W7 WI7 5 300 i f f H §§ m m I E 200 i J I I i I I strains DYI50 Acccl Acccl Rl DY1457ACCC/ &cccl W2 VV7 WI7 plasmid V V CCC1 V V V CCCI V V V FIGURE 2. Characteristics of high Iron-resistant Acccl mutants. A, DY150, DY150 A cccl Rl mutant, DY1457, DY1457 Acccl and W2, W7, and W17 mutants were grown In CM m e d i um overnight. 20 OD cells (2 x 10s) were collected, washed, and digested in nitric acid and iron contents were deter-mined by ICP. 8, strains were streaked o n t o CM plates containing 40 jlm BPS w i t h 2 and 10 (am FeS04 added back. Plates were i n c u b a t e d at 30 °Cfor 2 days. 1, Afet3;2, DY150;3 ,DY150AcccI;4, Rl mutant; 5 ,Met3;6, DY1457; 7, DY1457Acccl; 8, W2; 9, W7; 10, W17. C, cells as in A were g r o w n in CM medium, spotted o n t o CM or CM w i t h 0.005% H 2 0 2 or 2.5 mM paraquat. Plates were i n c u b a t e d at 30 °C for 2 days. D, cells as in A were transformed w i t h a reporter construct CCC7-lacZ and either a c o n t r o l vector (I/) or a low copy CCCI plasmid. The cells were g r o w n in CM-Leu-Ura medium to log phase, harvested, and p-galactosidase activity determined. The data were normalized for p r o t e i n c o n c e n t r a t i o n and error bars represent S.D. from three experiments. DECEMBER 5, 2008-VOLUME 283-NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33867 26 Metal Specificity of Vacuolar Transporters grow on iron-limited medium, which requires a functional iron transport system (Fig. 2B). These results suggest that the resist-ance to high iron cannot be explained only by decreased iron acquisition or increased iron export. If iron toxicity was due to increased oxidative damage resulting from Fenton chemistry, then increased antioxidant defenses might reduce such dam-age. To test this possibility we examined the ability of mutant cells to resist the effect of the oxidants H 2 0 2 and paraquat. One mutant (W17) showed a slight increase in paraquat resistance, but in general the mutants were as sensitive to oxidants as the parental strains (Fig. 2Q. We tested the possibility that the mutants were able to store or sequester cytosolic iron by measuring the expression of an iron-responsive reporter. Transcription of C.CC1 or a CCC1- lacZ reporter construct is increased by iron through activation of the transcription factor Yap5 (15). Wild type, Acccl, and A cccl mutants were transformed with a CCCl-lacZ construct and the effect of iron on induction of j3-galactosidase activity was determined. Compared with wild type cells, A cccl cells showed a higher expression of CCCl-lacZ, indicating the pres-ence of increased cytosolic iron. This induction of CCCl-lacZ was repressed when the CCC1 gene was introduced back to A cccl cells (Fig. 2D). Mutants W2 and W17 showed similar amounts of CCCl-lacZ activity compared with the parental Acccl strain. R1 and W7, however, showed lower levels of j3-ga-lactosidase compared with the parental strains. The R1 mutant was the most resistant to growth inhibition by high iron (cf. Fig. L4). Mutant R1 had a slight increase in cellular iron compared with Acccl but less cytosolic iron as indicated by the CCCl-lacZ reporter assay. This implies that the resistance to high iron growth might be due to its ability to sequester cytosolic iron. Taken together these results showed that different mechanisms may contribute to high iron resistance in mutant strains. The High Iron Resistance of Mutant R1 Is Caused by a Muta-tion in ZRCl-To identify the gene responsible for the high iron resistance of R1 a low copy genomic library was generated from R1 cells. The library was transformed into the Acccl cells and transformants able to grow on high iron were identified. Plasmids conferring resistance to high iron were isolated and the genes on the insert were identified. Several plasmids con-taining overlapping segments of chromosome XIII were iso-lated. ZRC1, a gene that encodes a vacuolar zinc transporter (5, 22), was identified as a common gene on all rescued plasmids. Subcloning of the insert showed that the R1 -specific ZRC\ plas-mid conferred iron resistance when transformed into Acccl cells (data not shown). Sequence analysis of R1-Z/?C1 revealed a mutation at position 131 of the coding sequence that altered an adenine to thymidine, which resulted in the substitution of isoleucine for asparagine at amino acid position 44 (referred to henceforth as ZRC1(N44I)). To confirm that this single amino acid change was responsible for high iron resistance, the aspar-agine at position 44 of the wild type Zrcl was changed to an isoleucine by site-directed mutagenesis. When transformed into Acccl cells, ZRC1 (N44I) was able to confer high iron resist-ance to Acccl cells (Fig. 3A). Zrcl was identified initially as a high copy suppressor of zinc-sensitive growth (5) and deletion of ZRC1 increases zinc sensi-tivity (7). Cells with a deletion in ZRC1 showed zinc-sensitive WT vcctor u r n r r r * i F f T B I l l ® I FIGURE 3.ZRC1 (N44II confers resistance to high Iron. A, wild type (W7) cells transformed w i t h empty vector and Accc/ transformed w i t h empty vector, ZRC1, ZRCHN44I), or ZRCHD4SA) were spotted onto CM-Ura plates w i t h or w i t h o u t 3 mM ferrous a m m o n i um sulfate. Plates were incubated at 30 °C for 2 days. 8, wild type and AzrcJ cells transformed w i t h empty vector ZRC1, ZRCH.N44I), or ZRCHD4SA) were spotted on CM-Ura plates w i t h or without 5 mMZnSOa . Plates were Incubated at 30 °C for 2 days. C, WT, Acccl Izrch or i c c c / A z r c / cells were spotted on CM w i t h or w i t h o u t 3 mM ferrous ammo-n i um sulfate or 5 mM ZnS04 . Plates were incubated at 30 °C for 2 days. growth that was suppressed by expression of plasmid contain-ing ZRC1 but not by a plasmid containing ZRCHN44I) (Fig. 3B). This result demonstrates that Zrcl(N44I) has lost its intrinsic zinc transport activity, and may explain why the R1 mutant is sensitive to high zinc concentration (data not shown). It is possible that a loss of function of ZRC1 protects Acccl from high iron toxicity by altering zinc homeostasis and mod-ulating the entire yeast transcriptome. To test this we generated a mutant ZRC1 based on the sequence alignment of Zrcl with the E. coli FieF, another member of the cation diffusion facili-tator family (23). The crystal structure of FieF suggests that Asp49 is involved in binding the zinc substrate. The homolo-gous residue in Zrcl is Asp45 and site-specific mutagenesis of ZRC1(D4SA) resulted in an expressed protein that was unable able to protect A zrcl cells from high zinc toxicity (Fig. 3 B). This construct was unable to permit Acccl cells to grow on high iron medium (Fig. 3A). Furthermore, deletion of ZRC1 in Acccl cells did not protect Acccl from high concentrations of iron (Fig. 3C). Together, these data suggest that loss of zinc transport activity is not sufficient to protect Acccl from high iron toxicity and also explains why mutant R1 shows dominant characteristics. Zrcl(N44I) Increased Vacuolar Iron-Because Zrcl localizes to the vacuolar membrane (9), it is possible that Zrcl(N44I) transports iron into the vacuole, which would decrease cytoso-lic iron. We tested this possibility by assaying the expression of a FET3-lacZ reporter. This reporter construct is regulated by the low iron sensing transcription factor Aftl (24). Overexpres- 3386 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 49 - DECEMBER 5, 2008 27 Metal Specificity of Vacuolar Transporters FET3-lacZ Vector Vector ZRC1 ZRCKN441) WT Acccl Vacuolar iron | 4 o £ c 2 Vector ZRCI ZRC1(N44I) FIGURE 4. ZRCHN44I) alters cytosolic Iron by transporting Iron Into the vacuole. A. wild type (WT) cells w i t h integrated FET3-lacZ were transformed w i t h empty vector, ZRCI, ZRCHN44I), or CCCI under their own promoters. Cells w e r e g r o w n to log phase a n d 0-galactosidase activity determined. S, WT cells w i t h empty vector, Acccl cells w i t h empty vector, ZRCI or ZRCHN44I) were transformed w i t h a CCCl-lacZ reporter construct. Cells were grown in CM medium to log phase and p-galactosidase activity determined. C, Acccl cells transformed w i t h a high copy empty vector, ZRCI or ZRCHN44I) under t h e ZRCI promoter were grown in CM medium w i t h 50 (iM FeS04 overnight. Vacuoles were isolated and iron content was determined by ICP. All data were normalized for protein concentrations and error bars represent S.D. from three experiments. sion of CCCI, by transporting iron into vacuoles, leads to lower cytosolic iron inducing the expression of a FET3-lacZ reporter (6) (Fig. 4A). Expression of ZRC1(N44I) was also able to induce FET3-lacZ activity compared with cells transformed with either a vector or wild type ZRCL Conversely, expression of ZRCl(N44l) but not wild type ZRCI was able to reduce the activity of a CCCl-lacZ reporter construct (Fig. 4B). These results confirm that expression of ZRC1(N44I) lowers cytosolic iron. To prove that Zrcl(N44I) transports iron into vacuoles, iron was measured in vacuoles isolated from A cccl cells trans-formed with empty vector or plasmids expressing ZRCI or ZRCI (N441). Overexpression of wild type ZRCI showed a slight increase in vacuolar iron relative to vector-transformed cells (Fig. 4C). Overexpression of ZRC1(N44I), however, resulted in a 3-4-fold increase in vacuolar iron. Together, these results show that ZRC1{N44I) is a gain of function allele that can con-fer iron-resistant growth by exporting iron from cytosol to vacuole. These data show that Zrcl(N441) can transport iron but it is hard to compare the intrinsic iron transport activity of Zrcl(N44I) with that of Cccl, as expression of each transporter is regulated differently. CCCI transcription and mRNA stabil-ity are increased by iron, whereas ZRCI (and by implication ZRC1(N44I)) shows increased transcription due to low zinc (7). To determine the intrinsic iron transport activity of Cccl, Zrcl, and Zrcl(N441), each was cloned into a GAL1 -regulated vector. We placed a His6 epitope at the carboxyl terminus of each gene, which permitted us to determine protein levels. GAL1 regu-lated CCCI-HISf, and ZRC1(N44I)-HIS6 were able to suppress the high iron growth defect of A cccl cells, whereas as a GAL1 regulated ZRC1-HIS6 was not (Fig. 5A). Under similar growth conditions, Zrcl-HIS,, and Zrcl(N44I)-HIS6 were expressed to higher levels than Cccl-HIS6 (Fig. 56). Measurement of iron transport activity based on FET3-lacZ expression showed that Zrcl-HIS6 had no measurable iron transport activity, whereas both Zrcl(N44I)-HISf, or Cccl-HISfl induced expression of FET3-lacZ (Fig. 5C). Based on the expression levels of the pro-teins Zrcl(N44I)-HIS6 has -10-15% of the iron transport activity of Cccl-HIS6. A cccl cells transformed with empty vec-tor or galactose-regulated constructs were grown in galactose medium with 100 /xM FeS04. Vacuoles were isolated and iron content determined. Overexpression of CCC1-HIS6 and ZRC1(N441)-H1S„, but not ZRC1-H1S6 increased vacuolar iron compared with vector-transformed control cells (Fig. 5D). There was more iron in Cccl-HIS6 vacuoles than in Zrcl(N44I)-HISfi vacuoles, again indicating that Cccl was more efficient in transporting iron into vacuoles. It might be possible that iron is not transported into the vac-uole but rather the iron is tightly bound to the cytosolic facing surface of Zrcl(N44I). The difference in vacuolar iron between A cccl cells overexpressing ZRC1{N441) and control cells (empty vector) was about 10-12 nmol/mg of vacuolar protein. The crystal structure of the E. coli CDF family member FieF suggests that it has 4 Zn binding sites (11). Assuming one mol-ecule of Zrcl(N44I) binds 4 atoms of iron, then based on the molecular mass of Zrcl (48,344 daltons), would require -0.12- 0.15 mg of Zrcl protein/mg of vacuole protein to bind the measured iron. This means that Zrcl(N44I) would have to con-stitute 12-15% of vacuolar proteins. To test this prediction we grew A cccl cells, transformed with either a control vector or G/lL-regulated ZRCKN44I) in galactose medium overnight and then in galactose medium containing 100 /u.m FeS04 for 4 h. Cells were harvested, vacuolar iron determined, and vacuolar proteins applied to SDS-PAGE and the gel analyzed by Western blot and silver stain. Vacuoles isolated from control cells only showed a slight increase in iron over the time course of the experiment, probably due to endocytosis of iron from the cul- DECEMBER 5, 2008-VLUME 283-NUMBER49 JOURNAL OF BIOLOGICAL CHEMISTRY 3386 28 Metal Specificity of Vacuolar Transporters CM-l!ra Gal 3 mM Fc 5 mM Fe Vacuolar iron WT Accc/ 34 - FET3-lacZ Vector ZRCI ZRCKN441) CCCI Vacuolar iron Oh I4h Vector ZRCI(N44I) Vector ZRC1(N441) hours anti-6XHIS anti-C'PY 0 0 4 Stds kDa 72 - 55 - 43 - Vector ZRC1(N44I) 0 4 0 4 hours Predicted size of ZrcI(N44l)-6XHIS 49 kDa Vector ZRCI ZRCI{N44I) CCCI FIGURE 5. Iron transport efficiency of Zrc1(N44l). A, wild t y p e (WT) cells transformed w i t h empty vector (pYES2 GALI promoter), Acccl cells trans-formed w i t h pYES2, ZRCI ZRCHN44I), or CCCI tagged with His6 at the car-boxyl terminus were g r o w n in CM w i t h 2% raffinose at 30 °C overnight. Cells were washed and spotted onto CM-Ura medium containing 2% galactose w i t h or w i t h o u t 3 or 5 mM ferrous a m m o n i um sulfate. Plates were incubated at 30 °C for 3 days. B, Acccl cells as in A were g r o w n in CM medium w i t h 2% raffinose t h e n inoculated into CM m e d i um w i t h 2% galactose for 12 h. Cells were harvested, disrupted by glass beads, and protein levels measured by Western blot using a rabbit anti-His6 antibody or a mouse anti-Vmal anti-body followed by peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. The vacuolar protein Vma1 was used as a loading control. C, Acccl cells w i t h an integrated FET3-lacZ at t h e HO locus were transformed w i t h empty vector pYES2,Z/?C/,ZRCl()V44/), or CCCI as in A Cells were g r o w n in CM medium w i t h 2% raffinoseand inoculated into CM w i t h 2% galactosefor 12 h. Cells were harvested and |3-galactosidase activity determined. The data are normalized for protein concentrations and error bars represent S.D. from three experiments. D, cells as in A were g r o w n in CM-Ura m e d i um containing 2% galactose w i t h 100 jxm FeS04 and 1 mM ascorbate overnight. Vacuoles were isolated and iron content was determined by ICP. All data were normal-ized for protein concentrations and error bars represent S.D. from three experiments. ture media. Vacuoles isolated from Zrcl(N441) showed a much greater increase in vacuolar iron (Fig. (>A). Western blot showed that during this time course, the Zrcl(N44I)-HIS6 protein was expressed and remained constant (Fig. 6B). Silver staining of vacuolar proteins from both control and Zrcl(N44I)-HlS6 expressing cells had a similar protein distribution in which the FIGURE 6. Measurement of vacuolar iron content and Zrcl (N44I) protein abundance. A, Accc 1 cells were transformed w i t h a control vector (pYES2) or a ZRC1{N44I)-HIS6 containing plasmid. Cells were g r o w n in galactose CM over-night and then incubated in t h e same m e d i um containing 100 jam FeSO„. Vacuoles were isolated from control and ZRCI(N44I) expressing cells at t i m e 0 and 4 h after incubation in iron-rich medium. The iron content of isolated vacuoles was determined by ICP and t h e data normalized to protein content, fi, isolated vacuoles were solubilized and analyzed by SDS-PAGE and Western blot using a rabbit anti-His6 a n t i b o d y or a mouse anti-carboxypeptidase Y antibody followed by peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. C, t h e same samples as in S were also analyzed by silver staining. The arrow represents t h e predicted mass of Zrcl (N44I)-HIS6. band corresponding to Zrcl(N44I)~HlS6 was not notable (Fig. 6Q, suggesting that the protein abundance would be insuffi-cient to bind vacuolar iron. Zrcl(N44I) Transports Manganese-Many transporters that transport iron also transport Mn2 + (25, 26). Deletion of CCCI results in sensitivity to high Mn2 + (27), although with the con-centrations and strain used here the sensitivity was slight at best. Expression of ZRC1{N44I) suppressed Mn2 4 toxicity in Accci cells to a greater extent than ZRCI (Fig. 7A). Pmrl is a Golgi membrane P-type ATPase involved in transporting Ca2 + and Mn2 + into the Golgi (28). Deletion of PMR1 leads to accu-mulation of Mn2 + in the cytosol, increasing the sensitivity of cells to high concentrations of Mn2 + . Indeed, CCCI was iden-tified as a high copy suppressor of Apmrl Mn2 1 toxicity phe-notype, indicating it also transports M n 2 + into vacuoles (27). 338 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 49 DECEMBER 5, 2008 29 Overexpression of CCCI or ZRC1(N44I), but not wild type ZRCI, protected Apmrl cells from Mn2 + toxicity (Fig. IB). These results indicate that Zrc(N44I) also has the ability to transport Mn2 + . To examine whether ZRC1(N44I) can transport copper into vacuoles, we studied the effect of over-expression of ZRC1(N44I) in copper-sensitive cells. ACE1 encodes a transcription factor that regulates the expression of metallothioneins, small intracellular proteins that can bind and detoxify copper (29). Deletion of ACE1 renders cells copper sensitive but overexpression of ZRCI or ZRC1(N44I) did not suppress or enhance the copper sensi-tivity of Aacel cells (Fig. 7C). A CM-Ura 15 mM Mn WT vcclor G Q L P • * / vcclor n o r I * • <* Acccl ZRCI n o c * • • r • 7.RCI(N44II n t ® A • • n •• B CM-l-eu 2 mM Mn I1Y4743 Apmrl /.KCI ZRCI(S'44I) UY4743 CCCI IRC! 7.RCKN441) FIGURE 7. ZRC1IN44I) shows an altered metal specificity. A, wild type (WD cells (DY150) transformed w i t h empty vector or Acccl cells transformed w i t h empty vector, ZRCI. or ZRCHN44I) were spotted o n t o CM-Ura plates, CM-Ura plates w i t h 15 mM MnCI2 . Plates were incubated at 30 °C for 3 days. B. w i l d type cells (BY4743) or Apmrl cells were transformed w i t h e m p t y vector, CCCI, ZRCI or ZRCHN44I) in a high copy vector w i t h their native promoter. Cells were spotted o n t o CM-Leu plates w i t h or w i t h o u t 2 mM MnCI2. Plates were incubated at 30 °C for 2 days. C, w i l d type cells (BY4743) or Aacel cells were transformed w i t h empty vector, CCCI, ZRCI or ZRCHN44I) in a high copy vector w i t h their native promoter. Cells were spotted o n t o CM-Leu plates w i t h or w i t h o u t 250 jam CuS04 . Plates were incubated at 30 °C for 2 days. I ScZrcl ScCr.tl H s Z n T - l MtoZnT-1 EcFieF M1TG-KELRIISLLTLDTVFFLLEITIGYMSH3LALIAD5FHMLNDIISLLVALWAV 56 ---MKLGSKOVKIISLLLLDTVFFGIEITTGYLSHSLAUADSFHMUfl;lISLVVAI.WAV 57 -HGCWGRNRGRLLCMLALTFMFMVI.EVWSRVTSSLAMLSDSFHMLSDVLALVVALVAE 58 --MGCWGRNRGRLLCMLLLTFMFMVLEWVSRVTASLAMLSDSFHMLSDVLALWAI.VAE 58 MNQSYGRLVSRAAIAATAHASLLLLIKIFAWWYTGSVSILAALVDSLVDIGASLTN1.LW 60 Metal Specificity of Vacuolar Transporters Cotl(N45I) Shows Increased Iron Transport and Decreased Cobalt Transport-The N44I mutation in Zrcl is in the second hydrophobic domain. The sequence of this domain is highly-conserved in homologous CDF proteins involved in Zn2 + transport, which are found throughout the biological kingdoms (Fig. 8). This sequence is highly conserved in the Zrcl homo-logue Cotl, which shows 78% amino acids similarity to Zrcl. Cotl was initially identified as a gene that conferred cobalt resistance when overexpressed (4). Cotl has Zn2 + transport activity, as overexpression of COT1 confers zinc resistance and deletion of COT1 increases zinc sensitivity in Azrcl cells (30). The corresponding asparagine at position 45 of Cotl was changed to isoleucine by site-directed mutagenesis to examine the effect on the substrate specificity of Cotl. Wild type COT1 or COTl(N45I) were transformed into A cccl cells and growth on high iron medium was examined. High copy expression of COT1 in Acccl cells was able to confer high iron resistance (Fig. 9A). Expression of C0T1(N45I) increased the resistance of Accci cells to high iron. Overexpressed COTl(N4SI) was able to induce FET3-lacZ reporter expression (Fig. 9B) and reduce expression of the CCCl-lacZ reporter (Fig. 9C) indicating an alteration of iron homeostasis. The N451 mutation, however, decreased the ability of COT1 to confer cobalt resistance on A cotl cells (Fig. 9 D). Overexpression of COT1 can partially suppress the high iron growth deficit of A cccl cells (Fig. 9A), suggesting that Cotl may be a low affinity iron transporter. In CM medium, however, expression of COT1 has little effect on Fet3-lacZ activity. COT1 is a target of the transcription factors Aftl and Aft2 (31) and shows a modest induction under low iron conditions. Low iron conditions prevent accumulation of the vacuolar iron trans-porter Cccl, as CCCI transcription is activated under high iron conditions (15) and CCCI mRNA is destabilized under low iron conditions by the Aftl-regulated gene CTH2 (14). We consid-ered the possibility that expression of a low affinity vacuolar iron transporter might protect cells against iron shock. Com-pelling evidence shows that expression of Zrcl by low zinc con-ditions protects cells against sudden increases in cytosolic zinc, termed zinc shock (7). We tested the possibility that Cotl might play an analogous role and protect cells from "iron shock." Cells grown in low iron were transferred to high iron medium and growth assayed. Wild type cells showed no obvious growth defect when incubated in high iron (Fig. 10). In contrast, A cccl cells showed a severe growth deficiency. Deletion of COT1 by itself did not affect growth in high iron nor did it exacerbate the growth defect of a CCCI deletion. These results do not support a role for Cotl in either iron shock conditions or in protecting cells from high iron. II FIGURE 8. Alignment of Zrcl homologues. The Zrcl protein sequence was used to BLAST search its homo-logues in other species. Sequences of selected homologues were retrieved and aligned w i t h ClustalW2. E. coli FieF, a CDF member, whose structure has already been determined, was also included In the alignment. The position of the mutated amino acids (N) In Zrcl and Cotl is indicated in italic. The t w o aspartate residues involved in zinc b i n d i n g in E. coli FieF are bolded. The predicated transmembrane domains are indicated. The abbreviations are: Sc, S. cerevisiae; Hs, Homo sapiens; Mm, Mus musculus-, Ec, E. coli K-12. Asterisk (*) denotes identity; colon (:) denotes similarity. DISCUSSION Deletion of the vacuolar iron transporter Cccl results in growth inhibition on high iron medium. We initiated this study to identify genes that suppressed the high iron growth defect. Our study identified both recessive and dominant muta- DECEMBER 5, 2008-VOLUME 283-NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 3387 30 Metal Specificity of Vacuolar Transporters CM-Ura 3 mM Fe l)Y 15<>-&<ccl-*~ Acoll Acccl Acotl FIGURE 9. COTHN45I) protects Acccl cells f r om high iron toxicity. A. wild type (WT) cells transformed w i t h a high copy empty vector and Acccl cells transformed w i t h empty vector, COT1, or C0THN45I) under the COT1 pro-moter were spotted o n t o CM-Ura plates w i t h or w i t h o u t 3 mM ferrous ammo-nium sulfate. Plates were incubated at 30 °Cfor 2 days. 8, WT cells containing an Integrated FET3-lacZ at the HO locus were transformed w i t h a high copy empty vector, C0T1, or C0THN4SI) under the COT! promoter. Cells were grown to log phase and /3-galactosidase activity determined. C, WT cells transformed w i t h a high copy empty vector, Acccl cells transformed w i t h empty vector, COT 1, or C0THN4SI) were transformed with a CCCl-lacZ reporter construct. Cells were g r o w n to log phase and p-galactosidase activ-ity determined. The data are normalized for protein concentrations and error bars represent S.D. from three experiments. D, wild t y p e cells (BY4743) trans-formed w i t h a high copy empty vector and Acotl cells transformed w i t h empty vector, COT1, or COTHN45I) under t h e COT) promoter were spotted o n t o CM-Ura w i t h or w i t h o u t 2 mM CoCI; . Plates were incubated at 30 °C for 2 days. tions that permitted A cccl cells to grow on high iron medium. The recessive genes fell into two complementation groups. We also identified eight dominant mutants, which were due to mutations in single genes, although genetic analysis did not permit us to determine whether the dominant mutants were in the same or different genes. Phenotypic analysis suggested that the dominant genes show differences in iron sensitivity and in their ability to modulate cytosolic iron levels (Figs. 1 and 2). 5 10 25 Fe((iM) FIGURE 10. c o n is not involved in iron shock. WT cells, Acccl, A c o t l . a n d Accc 1 A c o t l were g r o w n in CM m e d i um w i t h 40 /am BPS overnight. Cells were washed and inoculated into CM medium w i t h 40 /am BPS and specified con-centrations of FeS04 . Cell density was determined 12 h later. The experiment was performed multiple t i m es and a representative experiment is presented. Only one of the mutants showed notable resistance to oxidants suggesting that modulation of iron-dependent oxygen toxicity is not a prominent mechanism for high iron resistance. This result confirms our previous observation that high iron toxicity does not necessarily result from increased Fenton chemistry, as high iron growth deficit occurs anaerobically. We identified the molecular basis of one of the dominant iron-resistant mutants to be a single amino acid mutation in the vacuolar Zn2 + transporter Zrcl (22, 30). Targeted site-specific mutagenesis is the most common approach to examining the substrate specificity of enzymes or transporters. In combina-tion with either structural studies or genome wide informatics, targeted mutations have revealed much about the importance of specific domains or amino acids in determining substrate specificity. The role of amino acids in determining the substrate selectivity or transporters has been reported previously for such transporters, as the plant potassium transporter HI<T1 (32), H+-sucrose symporter AtSUCl from Arabidopsis thaliana (33), plasma membrane H 1 -ATPase PMA2 from Nicotiana plumbaginifolia (34), the plant A. thaliana ZIP family member metal transporter IRT1 (35), and yeast Saccharomyces cerevi-siae Mn2 + / C a 2 + transporter Pmrl (36). Amino acid substitu-tions generally abolish transporter activity or increase the transport activity of one of its substrates relative to another. Here we show that a single amino acid substitution in Zrcl or Cotl dramatically changed the substrate specificity. Genetic and biochemical studies have shown that Zrcl is able to trans-port Zn2 + , Ni2 + , Cd2 + , but not Fe2 + or Mn2 + (9). We con-firmed this conclusion by measuring resistance to high iron growth conditions and by reporter constructs that assay cyto-solic iron levels. Substitution of an asparagine at position 44 to an isoleucine abolished the ability of Zrcl to transport Z n 2 + but conferred the ability to transport Fe2 + and Mn2 + . A similar change in Cotl also altered its substrate specificity to iron while reducing its ability to transport Co2 + . Zrcl and Cotl belong to the CDF family of transition metal transporters. Members of this family are found in all biological kingdoms and most usually transport metals out of the cytosol either into organelles or out of cells. CDF transporters usually have six transmembrane regions in which there is a high degree of sequence conservation in the charged residues of transmem- 3 3 8 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 49 DECEMBER 5, 2008 31 brane domains II and V, which is thought to bind metals and form a transmembrane pore. The structure of the E. coli CDF member FieF, which transports iron, has been determined with Z n 2 1 in the metal binding site (23). Structural and mutagenesis studies using £. coli FieF have focused on the importance of the Asp4 5 and Asp4 9 in transmembrane TMD II, and H i s 1 5 3 and Asp'r " in TMD V as residues that coordinate Z n 2 4 in the crys-tal structure, and by implication iron in the native transporter. These charged residues are conserved in CDF family members although each family member shows different substrate speci-ficities. The mutated asparagine is adjacent to the conserved aspartic acid in TMD II of Zrcl and C o t l . The substitution of a hydrophilic residue with a hydrophobic residue may alter the conformation of the metal binding site, changing the metal specificity of transport. Our study suggests that t h e microenvi-roment of the amino acids adjacent to the metal binding aspar-tate determines the substrate selectivity. The mutated ZRCI was identified by a screen using random UV mutagenesis to select for cells showing high iron resistance. The approach of using a strong selection system in conjunction with mutagene-sis of genes encoding transporters offers the possibility of facile identification of other residues critical for determining the sub-strate specificity of CDF transporters. Acknowledgments-We express our appreciation to members of the Kaplan laboratory for critically reading the manuscript. REFERENCES 1. Ramsay, L. M„ and Gadd, G. M. (1997) FEMS Microbiol. Lett. 152, 293-298 2. Eide, D. J„ Bridgham, J. T„ Zhao, Z„ and Mattoon, J. R. (1993) Mol. Gen. Genet. 241, 447-456 3. Szczypka, M. S„ Zhu, Z„ Silar, P., and Thiele, D. ). (1997) Yeast 13, 1423-1435 4. Conklin, D. S„ McMaster, J. A.. Culbertson, M. R„ and Kung, C. (1992) Mol. Cell. Biol. 12, 3678-3688 5. Kamizono, A„ Nishizawa, M., Teranishi, Y., Murata, K„ and Kimura, A. (1989) Mol. Gen. Genet. 219,161-167 6. Li, L„ Chen, O. S„ McVey Ward, D„ and Kaplan, J. (2001) /. Biol. Chem. 276,29515-29519 7. MacDiarmid, C. W„ Milanick, M. A., and Eide, D. J. 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Chem. 275, 23933-23938 DECEMBER 5, 2008-VOLUME 283-NUMBER49 JOURNAL OF BIOLOGICAL CHEMISTRY 3387 CHAPTER 3 GAIN-OF-FUNCTION MUTATIONS IDENTIFY AMINO ACIDS WITHIN TRANSMEMBRANE DOMAINS OF THE YEAST VACUOLAR TRANSPORTER ZRCI THAT DETERMINE METAL SPECIFICITY 33 Biochem J (2009)422.273-283 (Printed in Great Britain) doi 101042/BJ20090853 2 7 3 Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrcl that determine metal specificity Huilan LIN*, Damali BURTON*', Liangtao LI*, David E. WARNER*, John D. PHILLIPSt, Diane McVEY WARD* and Jerry KAPLAN2 'Department ol Pathology, Department of Internal Medicine, School of Medicine, University of Utah, Salt Lake City, UT 84132, U.S.A., and fDivision of Hematology, Department of Internal Medicine, School of Medicine, University of Utah, Salt Lake City, UT 84132, U.S.A. Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn;+. Co!+, Fe: + , Cd:+, Ni: + and Mn:+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc I changed its substrate specificity from Zn;+ to Fe; + and Mir+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865-33873). Mutant Zrcl that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCCI) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRCI, we identified other amino acid substitutions within ZRC! that changed its metal specificity. All Zrc I mutants that transported Fe;+ could also transport Mn:+. Some Zrc I mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn:+. All of the amino acid substitutions that resulted in a gain in Fe: + transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal-binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains. Key words: cation diffusion facilitator, iron, manganese, transition metal, vacuole, yeast, zinc. INTRODUCTION Transition metal ions (Cu+/2+, Fe:+, Zn:+, Co:+ and Mir+) are essential for all organisms as they serve as cofactors for various proteins. They can also be toxic in excess owing to their participation in redox reactions or by their competing with other metals for protein binding sites. Organisms have evolved mechanisms to regulate uptake, delivery, storage and detoxification of these metals. One mechanism that prevents metal toxicity is metal transport out of the cytosol by CDF (cation diffusion facilitator) transporters. These transporters mediate metal resistance by either exporting metals out of cells or into intracellular organelles, thus reducing cytosolic metal concentration. Some examples include Escherichia coli ZitB [ I ] and mammalian ZnTl(Slc30al), which mediate zinc efflux from cells [2] and Saccharomyces cerevisiae Zrcl and Cotl, which transport zinc and cobalt into vacuoles [3,4], The majority of CDF transporters have six TMDs (trans-membrane domains) with the N- and C-termini extending into the cytosol. A recent study classified CDF family members into three major groups (Zn-+, Fe;+/Zn:+ and Mn;+) based on substrate specificity [5]. Structural studies of the putative Zn:+ transporters CzrB from Thermus thermophilic [6] and YiiP (also known as FieF) from E. coli [7] indicate that the metal-binding sites of the transporter are formed by charged residues in TMDs II and V. The importance of amino acids in determining substrate specificity has been examined by sequence comparisons of different transport groups and by site-specific mutagenesis. Mutations that abrogate metal transport provide little information on substrate selection, because such mutations may affect transport activity independently of substrate selection. For example, loss of function may be due to alteration of residues involved in metal binding, as demonstrated by the study of YiiP(DI57A) [7], or due to residues involved in stabilizing the protein structure, as implicated by the study of ZitB(E214A), CzcD(H237R) and CzcD(H280A) [8,9], In contrast, amino acid mutations that lead to a gain of function by changing the metal transported, by their very existence, indicate the involvement of that amino acid in substrate selection. In this regard, gain-of- function mutations are informative in identifying residues involved in substrate selection. We identified a mis-sense mutation in the yeast vacuolar Zn; + transporter Zrc I that resulted in the loss of Zn! + transport and the acquisition of Fe:+ and Mrr+ transport activity [10]. The finding that one amino acid substitution can change transport specificity provides strong evidence for a role of that residue in substrate selection. Mis-sense mutations that result in a gain, rather than a loss, of function provide a robust approach for examining substrate specificity in CDF transporters. We combined a strong selection, the ability of cells with a deletion in the gene that encodes the vacuolar iron transporter CCCI to grow on high iron medium, with random and directed mutagenesis of ZRCI to identify residues that alter the transport specificity of Zrc I. We identified gain-of-function mutations that give rise to altered metal specificity. Our studies show that amino acids near the putative metal-binding amino acids in TMD II, as well as amino acids in other TMDs that are distant from the metal-binding residues, are important in substrate selection. Abbreviations used: CDF, cation diffusion facilitator; CM-Ura, complete minimal mix minus uracil; CPY. carboxypeptidase Y; ICP. inductively coupled plasma; TMD, transmembrane domain. ' Present address; City Year San Jose/Silicon Valley 142 W. Santa Clara Street, San Jose, CA 95113, U.S.A. 2 To whom correspondence should be addressed (email [e-mail]). © The Authors Journal compilation © 2009 Biochemical Society 34 274 H. Lin and others Table 1 Primers used in the present study Mutations are shown in lower case. Primers Sequence Purpose Pri54 GATATGAAAGTAGTTGCATT Clone ZflCr Pri60 TTGGTACAGGAGGGAACAAG Clone ZflCl Pri86 CCTTCTTCAGATGGCCTTGA Sequence ZfiC) Pri87 CCCAAGGTTGGTTTTATACG Sequence ZflC) Pri88 TGCCCTTATCACGTAGAGCT Sequence ZflCl PR0599 AATATACCTCTATACTTTAACGTC Sequence ZflCJ Pri56 ATGATCACCGGTAAAGAATT Error-prone PCR PR0064 CGCCAGGGTTTTCCCAGTCACGAC Error-prone PCR Pril 15 CCATAGGTTATATGTCACATTCATTtGCCTTGATTGCCGATTC ZRC1(L33F) Pril 16 GAATCGGCAATCAAGGCaAATGAATGTGACATATAACCTATGG ZRC1(L33F) Pri94 GGCCTTGATTGCCGATTCATccCACATGTTGAATGATATC ZRC1(F40S) Pri95 GATATCATTCAACATGTGggATGAATCGGCAATCAAGGCC ZRC1(F40S) Pri64 GGCCTTGATTGCCGATTCATTTCACATGTTGATTGATATCATCTCTCTTTTAGTGGC ZRC1(N44I) Pri65 GCCACTAAAAGAGAGATGATATCAATCAACATGTGAAATGAATCGGCAATCAAGGCC ZRC1(N44I) Pri102 CACATGTTGAATGCTATCATCTCTCTTTTAGTGGCAC ZRC1(D45A) Pri103 GTGCCACTAAAAGAGAGATGATAGCATTCAACATGTG ZRCUD45A) Pril 17 GCCTTGATTGCCGATTCATcTCACATGTTGAlTGATATCATGTCTC ZRC1(F40S, N44I) Pril 18 GAGAGATGATATCAaTCAACATGTGAgATGAATCGGCAATCAAGGC ZRC1IF40S. N44I) Pri127 GCCGATTCATTTCACATGTTGgtTGATATCATCTCTCTTTTAGTGGC ZRCHN44V) Pri128 GCCACTAAAAGAGAGATGATATCAacCAACATGTGAAATGAATCGGC ZRC1(N44V) Pri129 GCCGATTCATTTCACATGTTGgcTGATATCATCTCTCTTTTAGTGGC ZRC1(N44A) Pri130 GCCACTAAAAGAGAGATGATATCAgcCAACATGTGAAATGAATCGGC ZRC1(N44A) Pri149 CATTGGCCTTGATTGCCGATTCAacTCACATGTTGAATGATATC ZRC1(F40T) Pri150 GATATCATTCAACATGTGAgtTGAATCGGCAATCAAGGCCAATG ZRC1(F40T) Pri151 CATTGGGCTTGATTGCCGATTCAgcTCACATGTTGAATGATATC ZRC1(F40A) Pri152 GATATCATTCAACATGTGAgcTGAATCGGCAATCAAGGCCAATG ZRC1(F40A) EXPERIMENTAL Yeast strains and growth media The following yeast strains (W303 background) were used: wild-type DY150 [Mat a ade2-l his3-ll leu2-3,112 trpl-1 ura3-52 canl-IOO(oc)]. A cccl and Azrcl strains were generated in the DY150 background by double-fusion PCR, using the HIS3 gene as a selectable marker as described in [11 J. Strains with a FET3- lucZ reporter integrated at the HO locus were constructed as described in [12]. Wild-type BY4743 (Mat a/a his3Al/his3Al leit2AO/leu2AO l\s2A0/+ metl5A0/+ ura3 AO/ura3 AO) and A pmrl ::KanMX strains in the B Y4743 background were obtained from Research Genetics. Yeast strains were grown in YPD medium (1 % yeast extract, 2% peptone and 2% dextrose) or in CM medium (0.67% yeast nitrogen base without amino acids, 2% dextrose and 0.13 % amino acid drop-out mix). Plates with high concentrations of metals were made by adding either ferrous ammonium sulfate, zinc sulfate or manganese chloride. Liquid media was supplemented with FeSOj to the indicated concentrations, with a final concentration of 1 mM ascorbic acid. Transposon mutagenesis ol Acccl A mTn-lacZ transposon insertion library from Professor Michael Snyder (Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, U.S.A.) was used for mutagenesis. Three separate library pools were digested with NotI and transformed into Acccl cells. Cells were replicate plated on to medium containing 3 mM iron, and mutants able to grow on high iron medium were selected for further study. The transposon tar get site was identified by sequencing as described in [13]. Error-prone PCR and isolation of mutants Wild-type ZRCI with its own promoter and 3' end was generated by PCR from genomic DNA using Pri54 and Pri60 as primers (Table 1). The PCR fragment was digested with Bglll and Kpnl and inserted into pRS426 (a yeast f/ZJAi-containing episomal vector) to generate ZRCJ1 pRS426. This plasmid was used as the template for error-prone PCR with PR0064 and Pri56 as pri-mers. Mutations were introduced by the PCR error of Taq DNA polymerase (Fisher Scientific). The PCR conditions were: 95 °C for 2 min, 35 cycles of (95 °C, 30 s: 55 °C, 30 s; 72 °C, 2 min) and 72°C for 10 min. Four independent PCR products were pooled from the first round of PCR and used to seed six independent PCRs for the second round of PCR. The PCR products from the second round were pooled and transformed together with a gap plasmid (Z/?C//pRS426 digested with Hindlll and Kpnl and the large fragment was purified) into Acccl cells. Cells were grown for two days and Ihen replica plated to CM-Ura (complete minimal mix minus uracil) plates containing 5 mM ferrous ammonium sulfate. Cells able to grow on high iron were selected for further study. Plasmids rescued from the high iron-resistant colonies were sequenced using primers pri86, pri87 and pri88, which covered the ZRCI coding region. Site-directed mutagenesis The generation of ZRCI/pYES2, a His„-tagged version of ZRCI under the control of the galactose-inducible promoter GAL1, was described previously [10]. Site-directed mutagenesis using ZRCI/pYES2 as a DNA template was performed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primers used in the present study are listed in Table I. All mutations were confirmed by DNA sequencing. © The Authors Journal compilation © 2009 Biochemical Society 35 Metal specificity of yeast transporter Zrcl 275 Western blot, jS-galactosidase activity and iron content Cells were disrupted with glass beads in the presence of protease inhibitors (1.0 mM PMSF, 10/(M pepstatin A and 20 ^M leupeptin) (Sigma). The supernatant from a low speed centrifugation was solubilized using SDS/PAGE sample buffer. Samples (20 ^g protein) were subjected to SDS/4-20% PAGE, transferred to nitrocellulose, and probed with rabbit anti-(Hisr, tag) (1:2000, Abeam) or mouse anti-CPY (carboxypeptidase Y) antibody (1:4(KM), Invitrogen), followed by peroxidase-con-jugated goat anti-rabbit or goat anti-mouse antibody (1:I0(X)0, Jackson ImmunoResearch). Membranes were developed using chemiluminescence reagents (PerkinElmer Life Sciences). /i-Galactosidase activity was performed in 96-well plates using o-nitrophenyl /1-galactoside as a substrate [12J. Protein concentration was determined by the bicinchoninic acid method (Pierce) using BSA as standard. Vacuoles were isolated as described previously in [14]. The iron content of isolated vacuoles was determined using a PerkinElmer ICP (inductively coupled plasma)-Optical Emission Spectrometer with a standard curve generated from mixed metal standards. Transposon insertion sites M u t a n t 2 2 * 3 u p s t r e am 1 3 15 hps M u t a n t 2 5 - 1 u p s t r e am - 3 0 4 hps Immunofluorescence Immunofluorescence was performed as described in [15]. Cells were incubated with a rabbit anti-(His„ tag) (1:100, Abeam) overnight at room temperature (20°C) in a humid chamber, and then incubated with an Alexa-594-conjugated goat anti-rabbit antibody (1:750; Invitrogen) for 2 h at room temperature. Figure 1 Identification of transposon-generated genomic mutants that protect Acccl cells from high iron toxicity (A) Wild-type (WT), Acccl and Acccl mutants 22-3 and 25-1 were grown on CM-Ura plates with 3mM ferrous ammonium sulfate. Plates were incubated at 30°C for 2 days. (B) The transposon insertion sites were identified by sequencing the rescued plasmids. The transposon was inserted upstream ol ZRC 1 in both mutants, as denoted by the arrow. RESULTS Random mutagenesis results in Zrcl mutations that have gained iron transport activity Zrcl is a yeast vacuolar metal transporter that protects cells from the toxic effect of high concentrations of Zn2+ [3,16]. We showed that a single amino acid change (N44I) in Zrcl dramatically altered its substrate specificity from Zn;+ to Fe2+ and Mir+ [10], As a consequence, expression of Zrcl (N44I) was able to rescue the high iron growth defect of Acccl cells by transporting and storing iron in the vacuoles. Zrcl(N44I) was identified through a UV-mutagenesis screen that selected for Acccl mutant cells able to grow on high iron medium. We also utilized a transposon-generated library [13] to identify high iron-resistant mutants of Acccl cells. From 15000 colonies, two of the transposon-containing mutants, 22-3 and 25-1, were able to grow on high iron (Figure 1 A). These mutants were found to have an insertion of the transposon upstream of ZRCI (Figure IB). The insertion point of the transposon in each mutant was different, demonstrating that they were independent events. The mutants, although iron resistant, were unable to grow on high Zn, suggesting an alteration in Zrcl substrate specificity (results not shown). Sequencing of ZRCI in each of the mutants revealed base changes resulting ill an amino acid substitution, although different in each case. Mutant 22-3 showed an A to T nucleotide change at position 131, resulting in a N44I amino acid substitution in Zrcl. The second mutant, 25-1, showed a G to T nucleotide change at position 99, resulting in a L33F amino acid substitution in Zrcl. Thus two different ZRCI mutations lead to a high iron-resistant phenotype. The topology of Zrc I and the location of the amino acid substitutions within Zrc 1 are shown in Figure 2. Other mutations in Zrcl that resulted in a change in metal substrate specificity were discovered through a second genetic screen. Previously, we identified MRS3 and MRS4 as high-copy suppressors of iron toxicity in a Acccl strain [17]. To identify other high-copy suppressors of poor growth of Acccl on high iron and circumvent the identification of MRS3 and MRS4, we gene-rated a high-copy genomic library from Amrs3Amrs4 cells. The genomic library was transformed into Acccl cells, and cells cap-able of growth on high iron medium were selected. As expected, we identified plasmids containing CCCI (identified by colony PCR) as suppressors that conferred high iron resistance (results not shown). We also identified two high copy suppressors that contained ZRCI with T to C nucleotide mutations at positions 119 and 120, resulting in the same mis-sense mutation F40S in Zrcl. Directed evolution ol Zrcl reveals that diflerent mutations lead to acquisition of iron transport activity To identify other Zrc I mutants that were able to transport iron, the ZRCI coding sequence was randomly mutagenized through error-prone PCR. The PCR products were transformed together with a gap plasmid into Acccl cells. We performed PCR mutagenesis on two independent preparations of ZRCI. Transformed colonies were replica plated on to medium containing 5 mM ferrous ammonium sulfate. Colonies (50 000) were screened and resistant colonies (250) were identified. Plasmids from 62 resistant colonies were rescued, their ability to support the growth of Acccl cells reconfirmed and rescued plasmids were sequenced. Many mutant ZRCI plasmids showed more than one base change, but often the second base change was silent, as it did not result in a change in amino acid sequence (Table 2). We identified a number of mu-tants that had the same amino acid substitution (e.g. F40S). We know that those mutants were independently generated, as some mutants had a silent base change in addition to the mis-sense mutation (compare pZml65 with pZm225). We also identified the same amino acid substitution occurring by itself and in conjunction with other amino acids (compare pZml65 with pR3 and pR26). We also identified different substitutions of the same amino acid (L33S/L33F and I275N/I275F). These observations © The Authors Journal compilation © 2009 Biochemical Society 36 27 H. Lin and others Vacuole Cytoplasm Figure 2 Model for the topology of Zrcl A model for the topology of Zrcl is shown with predicted TMDs in the centre (I to VI), cytoplasmic loops below and vacuolar loops above. Black circles are residues in TMDs II and V that co-ordinate zinc based on the homologous bacterial CDF transporter YiiP. The numbered grey circles are residues identified in the present study that, when mutagenized, result in iron transport activity. The histidine-rich and cytosolic domains are also indicated. Transmembrane domains were predicted using the TMHMM program. suggest that the mutagenesis was extensive; however, as shown below, we do not think it was saturated. We first focused on plasmids in which there was only one amino acid substitution. All the ZRCI mutants were able to protect Acccl cells from high iron toxicity (Figure 3A). For comparison purposes we included ZRC1(D45A), which we had generated previously [IO], Aspartic acid in the corresponding position in the E. coli YiiP is a critical zinc-binding residue [7]. Zrcl(D45A) does not have Zn:+ or Fe:+ transport activity. To further demonstrate that the mutant proteins altered cellular iron metabolism, they were overexpressed in wild-type cells containing an iron-sensitive reporter FET3-lacZ. Overexpression of Cccl, by transporting iron into the vacuoles, lowers cytosolic iron, resulting in induction of FET3-lacZ [14]. Overexpression of wild-type Zrcl did not change the expression level of this reporter; however, all Zrcl mutants were able to induce FET3- IcicZ, indicating their ability to lower cytosolic iron, although less efficiently than Cccl (Figure 3B). Consistent with loss of trans-port activity, Zrcl(D45A) had no effect on expression of FET3- IcicZ. Previously, we demonstrated that Zrc l (N44I) protects Acccl from high iron toxicity by transporting iron into vacuoles [10]. Overexpression of selected mutant Zrcl proteins increased vacuolar iron content in Acccl cells relative to cells transformed with a vector control or with wild-type ZRCI (Figure 3C). Two amino acid substitutions in Zrcl are less efficient in transporting iron than a single amino acid substitution To test whether combining amino acid substitutions might result in an additive effect generating a more efficient Fe:+ transporter, three independently isolated mutants in TMD II (L33F. F40S and N441) were chosen to generate double mutants. Zrcl with two amino acid substitutions was not more efficient in Fe2+ transport activity than Zrcl with a single substitution, as assayed by growth on high iron medium (Figure 3A) and induction of FET3-lacZ (Figure 4A). We note that Zrcl(L33F, F40S) showed a low induction of FET3-lacZ, yet conferred high iron resistance when assayed on plates (Figure 3A). This difference may be ascribed to a difference in affinity, as the induction of FET3- lacZ was performed in moderate iron medium, whereas the plate assay utilized high iron medium. Zrc l (L33F, N44I) conferred less protection from high Fe2+ toxicity than the single substitution mutants, and its ability to induce the low iron sensor FET3-lacZ was also reduced. It is possible that some of the double amino acid substitutions give rise to an unstable Zrcl. To test this, wild-type Zrcl, single mutants and double mutants were epitope tagged at the C-terminus with His,, and expressed using the GAL1 promoter. The expression levels of single mutants and double mutants were similar to that of the wild-type Zrc 1 (Figure 4B). We also tested the possibility that the double-mutant proteins were mistargeted and did not accumulate in the vacuole. Immunofluorescence showed that all Zrcl mutants were targeted to the vacuole (Figure 4C). These data suggest that combining some amino acid substitutions in Zrc I did not have an additive effect on iron transport. Gain ol iron transport activity in Zrcl mutants does not necessarily mean the loss of intrinsic zinc transport activity As described previously, Zrc I (N44I) gained Fe2+ transport activ-ity but lost its inherent Zn:+ transport activity [10], Loss of Zn: + transport is not, however, sufficient to protect Acccl from high concentrations of iron. Zrcl(D45A) does not have Zn2+ transport activity and does not protect Acccl cells from high iron [10], In The Authors Journal compilation © 2009 Biochemical Society 37 Table 2 Nucleotide and amino acid changes in mutant Zrcl Mutants Amino acid change Nucleotide change pR3 F40S.G410D t119c. g!229a pR4 L33F g99t pR8 F40S. silent 1119c. a1245g pR9 N44I, P371L a131t, C1112I pR10 A52V. I94T, V239I. L273F, C1551,1281c, g715a,a819t pR13 N44I,194T a131t, 1281c pR15 A37G, silent, silent c110g,t726c,t1284c pRI6 I275F a823t pR17 L33F. silent g99t, 1628c pR18 Silent. I275N 1783c. t824a pR19 F40S t119c pR20 Silent. I177F, I275F. I387T a321a.a529t.a823t.t1163c pR22 F40S 1119c pR26 F40S, I94T t119c. 1281c pR28 I275N t824a pR31 F40S,S403G 1119c, a1207c pR32 Silent. I94T, G248C, I274V a90l, t281c. g742t. a820g pR124 N44I. I270T a131t. t809c pR127 Silent, F40S, D342E. silent, silent t54a. t119c. c1026g. 11248c. a1275t pR129 F40S, silent. I94T. silent t119c. 1237c. t281c,t1281c pRI30 F86S. I94T.D173G. silent, silent, I337T t257c.t281c, a518g. 1804c. a903g. a990g,t1010c pR132 N44T, silent. T259A a131c, t342c. a775g pR133 K224R, I275N, silent a671g, t824a, c948a pR134 L33F.V58A g99t. t173c. pR137 F40S. I94T. I177N. I388F t119c.t281c,I530a.a1162t pR139 I275N 1824a pR145 L33F g99t pZm152 F19V. F40S.S222P t55g,t119c, 1664c pZm155 I275N t824a pZm160 F40S t119c pZm163 E97G, silent a290g. t795c pZm165 F40S t119c pZm167 L33S. S166P, Y419C I98c,t496c. a1256g pZm169 L87H t260a pZm!71 L87H,S387P t260a. t1159c pZm175 E104G.N231S a311g,a692g pZm!76 E97G, silent, silent a290g,t831c, t856c pZml77 L33S, silent t98c,a717g pZm184 A52T, silent g154a. 11191c pZm185 Low quality but has A52T g154a pZm187 L87H t260a pZm188 E97G a290g pZm192 L33S. silent I98c.t583c pZm194 E97G a290g pZm195 F20L, silent, P105L 158c, t81c, c314t pZm207 L33S 198c pZm208 Silent. S272P c516t. t814c pZm210 E97G,E107D a290g.a321t pZm211 R101G, silent a301g.a939g pZm213 R101G, silent. F401L a301g. a903g.11203a pZm214 M232T, S272P t695c. t814c. pZm217 E97G. silent. E179G a290g.t417c. a536g pZm218 L33F g99t pZm219 I36N, silent, L203S, E399A I107a,t171c, I608c,a1196c pZm223 G79S g235a pZm224 G79S g235a pZm225 F40S, silent 1119c. t309c pZm226 S272P 1814c pZm229 Silent. S272P a564c,t814c pZm230 F86S t257c pZm235 P269L. S272P c806t. t814c pZm236 F40S.T276S t119c, c827g our previous study [10], we used a low copy plasmid to test Zn2+ transport activity of mutant Zrc 1. In the present study we used a high copy plasmid to determine if overexpressed mutant Zrcl retained any Zn:+ transport activity, as assayed by protection of Metal speciiciy of yeast transporter Zrcl 2 Azrcl cells from high zinc toxicity. Some mutants (L33F and L87H) clearly lost Zn:+ transport activity, however, others (F40S, A52T, G79S, F86S, R101G and I275F) did not show significant differences from the wild-type Zrcl in protecting Azrcl cells from zinc toxicity (Figure 5). Other mutants of Zrcl (L33S, N44I, S272Pand I275N) showed decreased Zn:+ transport activity compared with wild-type Zrcl. It is interesting that the double mutant Zrcl(F40S, N44I) lost Zn2+ transport activity, whereas individually each of the single mutants had Zn:+ transport activity. Zrcl mutants that transport iron also transport manganese The Golgi membrane P-type ATPase Pmrl is responsible for transporting Ca;+ and Mir+ into the Golgi [16]. Apmrl cells accumulate Mn:+ in the cytosol and show poor growth on high concentrations of Mir+ [17], Overexpression of Cccl, by transporting Mn:+ into the vacuoles, protects Apmrl cells from toxic amounts of manganese in the environment. To test whether the Zrcl mutants were able to transport Mn:+ into vacuoles and detoxify it, Apmrl cells were transformed with these mutant con-structs and grown on plates with 3 mM Mir4". All the mutants showed protection of Apmrl cells compared with wild-type Zrc 1 (Figure 6), although some mutants were less efficient than others. Surprisingly, although the D45A substitution lost both Fe:+ and Zn2' transport activity, it was able to partially rescue the growth defect of Apmrl. Effect of amino acid substitutions in TIVI2 on substrate selection The sequence adjacent to the putative metal-binding residues in TMD II is different in different CDF family members. These amino acids were hypothesized to determine the substrate specificity of CDF families. We identified two residues, PheJ" and Asn44, adjacent to the putative metal-binding residues His41 and Asp45 in TMD II, which, when mutated, resulted in iron transport activity (F40S and N44I). The F40S mutation occurred quite often and no other mutations of Phe4" were found. One interpretation of this result is that the hydroxy group of the introduced serine residue is required for Fe:+ transport. To test this possibility, we determined whether other substitutions in this amino acid would affect substrate selection. We generated Zrcl(F40T) and Zrcl(F40A) and examined their effect on metal resistance. Zrcl(F40S) and Zrcl(F40T) were able to protect Acccl cells from high iron toxicity (Figure 7A). Complementation by F40A was less efficient than for F40S or F40T, but was still noticeable (note vector alone). This result indicates that the hydroxy group at position 40 enhances, but is not absolutely required for, Fe;+ transport. All the Phe4" mutants retained Zn;+ transport activity, with Zrcl(F40S) having slightly decreased activity (Figure 7B). Zrcl(F40S) pro-tected Apmrl cells from Mn;+ toxicity to a much greater extent than F40T (Figure 7C). The fact that F40T did not lead to Mn!+ transport shows that it is not simply the presence of a hydroxy group that permits transport. A hydrophobic amino acid substitution in Zrcl at position 44 (N44I) resulted in Fe:+ transport activity. This residue is adjacent to the putative metal-binding residue Asp45 and may alter the structure of the metal-binding site. To examine whether the size of the side chain at this position affects substrate selection, we generated N44I, N44A and N44V mutations. All Asn44 substitutions were able to protect Acccl cells from iron toxicity (Figure 7A). Zrc 1 (N44A) retained Zn:+ transport activity, whereas Zrcl(N44I) and Zrcl(N44V) did not (Figure 7B). All three mutants were able to transport Mn:+, as shown by their © The Authors Journal compilation © 2009 Biochemical Society 38 27 H. Lin and others WT pRS426 pRS426 ZRCI ZRC1(L33F) ZRC I (U.IS) ZRCKF40S) ZRC1(N441) ZRC1(A52T) ZRCKG79S) ZRCKFH6S) ZRCHL87HI ZRCI(E97G) ZRCI(RIOIG) ZRCI(S272P) ZRCHI275F) ZRC1U275N) ZRC1(D45A) ZRCI(L33F. F40S) ZRCHU3F. N44I) ZRCKF40S. N44D CM-Ura 5 mM Fc B i r u i x i : n i i i r LI I'M! [ 1 M B H I M l l l f f E-t u r n L i l l l i ULKJC Li I B a N c u b i i n • • * • GOOD • E D S m L • • • * LULE ULKB r r z z OTEJB I I I t l C • o r i s ULU3 m i a m n • • * l 1 1 CI • * ' l i t ) r m p |i-galactosidasc activity (nraol/min/mg prolcin) 0 10 20 .10 40 pRS426 | ZRCI L33F L33S F40S N441 A52T G79S KS6S I.X7H H97G RIOIG S272P 1275F I275N D45A 0 pRS426 • 1C1C C-/ I i l__ 2(1 4(1 60 X0 1110 Vacuolar iron pRS42G ZRCI L33F N44I I275F Figure 3 Zrcl mutants protect Acccl cells from high iron toxicity (A) DY150 (WT) and Acccl ceils, transformed with Ihe indicated plasmids, were grown in CM-Ura medium to exponential phase and serially spotted on to CM-Ura plates with or without 5 mM lerrous ammonium sulfate. The plates were incubated at 30°C for 2 days, (B) WT cells with an integrated FET3-lacZ reporter construct were transformed with pRS426 (empty vector), ZRC1. ZRC1 mutants and CCC1 regulated by their endogenous promoters. Cells were grown to exponential phase in CM medium and /)-galaclosidase activity was determined, (C) A cccl cells transformed with pRS426, ZRC1 or mutant ZRC1 under the control of the ZRC1 promoter and were grown in CM-Ura medium with 100 /iM FeS0< overnight. Vacuoles were isolated and Ihe iro