Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity

Yeast strains and growth media

The following yeast strains (W303 background) were used: wild-type DY150 [Mat a ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100(oc)]. Δccc1 and Δzrc1 strains were generated in the DY150 background by double-fusion PCR, using the HIS3 gene as a selectable marker as described in [11]. Strains with a FET3-lacZ reporter integrated at the HO locus were constructed as described in [12]. Wild-type BY4743 (Mat a/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/+ met15Δ0/+ ura3Δ0/ura3Δ0) and Δpmr1::KanMX strains in the BY4743 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 FeSO₄ to the indicated concentrations, with a final concentration of 1 mM ascorbic acid.

Transposon mutagenesis of Δccc1

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 Δccc1 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 target site was identified by sequencing as described in [13].

Error-prone PCR and isolation of mutants

Wild-type ZRC1 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 BglII and KpnI and inserted into pRS426 (a yeast URA3-containing episomal vector) to generate ZRC1/pRS426. This plasmid was used as the template for error-prone PCR with PR0064 and Pri56 as primers. 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 (ZRC1/pRS426 digested with HindIII and KpnI and the large fragment was purified) into Δccc1 cells. Cells were grown for two days and then 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 ZRC1 coding region.

Site-directed mutagenesis

The generation of ZRC1/pYES2, a His₆-tagged version of ZRC1 under the control of the galactose-inducible promoter GAL1, was described previously [10]. Site-directed mutagenesis using ZRC1/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 1. All mutations were confirmed by DNA sequencing.

Western blot, β-galactosidase activity and iron content

Cells were disrupted with glass beads in the presence of protease inhibitors (1.0 mM PMSF, 10 μM pepstatin A and 20 μM leupeptin) (Sigma). The supernatant from a low speed centrifugation was solubilized using SDS/PAGE sample buffer. Samples (20 μg protein) were subjected to SDS/4–20% PAGE, transferred to nitrocellulose, and probed with rabbit anti-(His₆ tag) (1:2000, Abcam) or mouse anti-CPY (carboxypeptidase Y) antibody (1:4000, Invitrogen), followed by peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody (1:10000, Jackson ImmunoResearch). Membranes were developed using chemiluminescence reagents (PerkinElmer Life Sciences). β-Galactosidase activity was performed in 96-well plates using o-nitrophenyl β-galactoside as a substrate [12]. 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.

Immunofluorescence

Immunofluorescence was performed as described in [15]. Cells were incubated with a rabbit anti-(His₆ tag) (1:100, Abcam) 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.

Random mutagenesis results in Zrc1 mutations that have gained iron transport activity

Zrc1 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 Zrc1 dramatically altered its substrate specificity from Zn²⁺ to Fe²⁺ and Mn²⁺ [10]. As a consequence, expression of Zrc1 (N44I) was able to rescue the high iron growth defect of Δccc1 cells by transporting and storing iron in the vacuoles. Zrc1 (N44I) was identified through a UV-mutagenesis screen that selected for Δccc1 mutant cells able to grow on high iron medium. We also utilized a transposon-generated library [13] to identify high iron-resistant mutants of Δccc1 cells. From 15000 colonies, two of the transposon-containing mutants, 22-3 and 25-1, were able to grow on high iron (Figure 1A). These mutants were found to have an insertion of the transposon upstream of ZRC1 (Figure 1B). 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 Zrc1 substrate specificity (results not shown). Sequencing of ZRC1 in each of the mutants revealed base changes resulting in 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 Zrc1. The second mutant, 25-1, showed a G to T nucleotide change at position 99, resulting in a L33F amino acid substitution in Zrc1. Thus two different ZRC1 mutations lead to a high iron-resistant phenotype. The topology of Zrc1 and the location of the amino acid substitutions within Zrc1 are shown in Figure 2.

Other mutations in Zrc1 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 Δccc1 strain [17]. To identify other high-copy suppressors of poor growth of Δccc1 on high iron and circumvent the identification of MRS3 and MRS4, we generated a high-copy genomic library from Δmrs3Δmrs4 cells. The genomic library was transformed into Δccc1 cells, and cells capable of growth on high iron medium were selected. As expected, we identified plasmids containing CCC1 (identified by colony PCR) as suppressors that conferred high iron resistance (results not shown). We also identified two high copy suppressors that contained ZRC1 with T to C nucleotide mutations at positions 119 and 120, resulting in the same mis-sense mutation F40S in Zrc1.

Directed evolution of Zrc1 reveals that different mutations lead to acquisition of iron transport activity

To identify other Zrc1 mutants that were able to transport iron, the ZRC1 coding sequence was randomly mutagenized through error-prone PCR. The PCR products were transformed together with a gap plasmid into Δccc1 cells. We performed PCR mutagenesis on two independent preparations of ZRC1. Transformed colonies were replica plated on to medium containing 5 mM ferrous ammonium sulfate. Colonies (50000) were screened and resistant colonies (250)were identified. Plasmids from 62 resistant colonies were rescued, their ability to support the growth of Δccc1 cells reconfirmed and rescued plasmids were sequenced. Many mutant ZRC1 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 mutants 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 pZm165 with pZm225). We also identified the same amino acid substitution occurring by it self and in conjunction with other amino acids (compare pZm165 with pR3 and pR26). We also identified different substitutions of the same amino acid (L33S/L33F and I275N/I275F). These observations 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 ZRC1 mutants were able to protect Δccc1 cells from high iron toxicity (Figure 3A). For comparison purposes we included ZRC1(D45A), which we had generated previously [10]. Aspartic acid in the corresponding position in the E. coli YiiP is a critical zinc-binding residue [7]. Zrc1 (D45A) does not have Zn²⁺ or Fe²⁺ 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 Ccc1, by transporting iron into the vacuoles, lowers cytosolic iron, resulting in induction of FET3-lacZ [14]. Overexpression of wild-type Zrc1 did not change the expression level of this reporter; however, all Zrc1 mutants were able to induce FET3-lacZ, indicating their ability to lower cytosolic iron, although less efficiently than Ccc1 (Figure 3B). Consistent with loss of transport activity, Zrc1 (D45A) had no effect on expression of FET3-lacZ. Previously, we demonstrated that Zrc1 (N44I) protects Δccc1 from high iron toxicity by transporting iron into vacuoles [10]. Overexpression of selected mutant Zrc1 proteins increased vacuolar iron content in Δccc1 cells relative to cells transformed with a vector control or with wild-type ZRC1 (Figure 3C).

Two amino acid substitutions in Zrc1 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 Fe²⁺ transporter, three independently isolated mutants in TMD II (L33F, F40S and N44I) were chosen to generate double mutants. Zrc1 with two amino acid substitutions was not more efficient in Fe²⁺ transport activity than Zrc1 with a single substitution, as assayed by growth on high iron medium (Figure 3A) and induction of FET3-lacZ (Figure 4A). We note that Zrc1 (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. Zrc1 (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 Zrc1. To test this, wild-type Zrc1, single mutants and double mutants were epitope tagged at the C-terminus with His₆ and expressed using the GAL1 promoter. The expression levels of single mutants and double mutants were similar to that of the wild-type Zrc1 (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 Zrc1 mutants were targeted to the vacuole (Figure 4C). These data suggest that combining some amino acid substitutions in Zrc1 did not have an additive effect on iron transport.

Gain of iron transport activity in Zrc1 mutants does not necessarily mean the loss of intrinsic zinc transport activity

As described previously, Zrc1 (N44I) gained Fe²⁺ transport activity but lost its inherent Zn²⁺ transport activity [10]. Loss of Zn²⁺ transport is not, however, sufficient to protect Δccc1 from high concentrations of iron. Zrc1 (D45A) does not have Zn²⁺ transport activity and does not protect Δccc1 cells from high iron [10]. In our previous study [10], we used a low copy plasmid to test Zn²⁺ transport activity of mutant Zrc1. In the present study we used a high copy plasmid to determine if overexpressed mutant Zrc1 retained any Zn²⁺ transport activity, as assayed by protection of Δzrc1 cells from high zinc toxicity. Some mutants (L33F and L87H) clearly lost Zn²⁺ transport activity, however, others (F40S, A52T, G79S, F86S, R101G and I275F) did not show significant differences from the wild-type Zrc1 in protecting Δzrc1 cells from zinc toxicity (Figure 5). Other mutants of Zrc1 (L33S, N44I, S272P and I275N) showed decreased Zn²⁺ transport activity compared with wild-type Zrc1. It is interesting that the double mutant Zrc1 (F40S, N44I) lost Zn²⁺ transport activity, whereas individually each of the single mutants had Zn²⁺ transport activity.

Zrc1 mutants that transport iron also transport manganese

The Golgi membrane P-type ATPase Pmr1 is responsible for transporting Ca²⁺ and Mn²⁺ into the Golgi [16]. Δpmr1 cells accumulate Mn²⁺ in the cytosol and show poor growth on high concentrations of Mn²⁺ [17]. Overexpression of Ccc1, by transporting Mn²⁺ into the vacuoles, protects Δpmr1 cells from toxic amounts of manganese in the environment. To test whether the Zrc1 mutants were able to transport Mn²⁺ into vacuoles and detoxify it, Δpmr1 cells were transformed with these mutant constructs and grown on plates with 3 mM Mn²⁺. All the mutants showed protection of Δpmr1 cells compared with wild-type Zrc1 (Figure 6), although some mutants were less efficient than others. Surprisingly, although the D45A substitution lost both Fe²⁺ and Zn²⁺ transport activity, it was able to partially rescue the growth defect of Δpmr1.

Effect of amino acid substitutions in TM2 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, Phe⁴⁰ 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 Phe⁴⁰ were found. One interpretation of this result is that the hydroxy group of the introduced serine residue is required for Fe²⁺ transport. To test this possibility, we determined whether other substitutions in this amino acid would affect substrate selection. We generated Zrc1 (F40T) and Zrc1 (F40A) and examined their effect on metal resistance. Zrc1 (F40S) and Zrc1 (F40T) were able to protect Δccc1 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, Fe²⁺ transport. All the Phe⁴⁰ mutants retained Zn²⁺ transport activity, with Zrc1 (F40S) having slightly decreased activity (Figure 7B). Zrc1 (F40S) protected Δpmr1 cells from Mn²⁺ toxicity to a much greater extent than F40T (Figure 7C). The fact that F40T did not lead to Mn²⁺ transport shows that it is not simply the presence of a hydroxy group that permits transport.

A hydrophobic amino acid substitution in Zrc1 at position 44 (N44I) resulted in Fe²⁺ 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 Δccc1 cells from iron toxicity (Figure 7A). Zrc1 (N44A) retained Zn²⁺ transport activity, whereas Zrc1 (N44I) and Zrc1 (N44V) did not (Figure 7B). All three mutants were able to transport Mn²⁺, as shown by their ability to protect Δpmr1 cells from Mn²⁺ toxicity (Figure 7C). A summary of phenotypes for all mutant Zrc1 isoforms is detailed in Table 3.