Transmembrane Segment XI of the Na+/H+ Antiporter of S. pombe is a Critical Part of the Ion Translocation Pore

The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes intracellular sodium in exchange for an extracellular proton. We examined the structure and functional role of amino acids 360–393 of putative transmembrane (TM) segment XI of SpNHE1. Structural analysis suggested that it had a helical propensity over amino acids 360–368, an extended region from 369–378 and was helical over amino acids 379–386. TM XI was sensitive to side chain alterations. Mutation of eight amino acids to alanine resulted in loss of one or both of LiCl or NaCl tolerance when re-introduced into SpNHE1 deficient S. pombe. Mutation of seven other amino acids had minor effects. Analysis of structure and functional mutations suggested that Glu361 may be involved in cation coordination on the cytoplasmic face of the protein with a negative charge in this position being important. His367, Ile371 and Gly372 were important in function. Ile371 may have important hydrophobic interactions with other residues and Gly372 may be important in maintaining an extended conformation. Several residues from Val377 to Leu384 are important in function possibly involved in hydrophobic interactions with other amino acids. We suggest that TM XI forms part of the ion translocation core of this Na+/H+ exchanger.

higher eukaryotes, are not well studied. In the well studied Na + /H + exchangers, one key feature that has been noted is the critical nature of transmembrane (TM) segments IV and XI. These two segments create a characteristic fold. Both are discontinuous helices and at their crossing point are unfolded, accommodating charged and polar residues that neutralize the dipoles of the helices within the lipid bilayer. This region may harbor the ion translocation center 11,14 . This fascinating arrangement and the details of these transmembrane segments have not been well demonstrated in other Na + /H + exchangers of higher species such as in vertebrate, plant or yeast Na + / H + exchangers.
In this study, we examined the structure and function of TM XI of SpNHE1. We used alanine scanning mutagenesis to characterize functional residues of this region of the protein. Additionally, we examined the structure of this TM segment by NMR spectroscopy. This region appears similarly structured to other Na + /H + exchangers, with a putative helix-extended region-helix in dodecylphosphocholine (DPC) micelles. A number of regions of the TM segment are important in function, possibly contributing to cation coordination or maintaining structure of this TM segment or in interaction with other transmembrane segments. Our results are the first examination of the structure and function of this region of the membrane protein. They support the hypothesis that SpNHE1 has a "Na + /H + exchanger"-like fold within the protein and that amino acids 360-393 are a critical part of this fold.

Results
SpNHE1 alignment and modeling. We examined amino acids of the putative TM segment XI of SpNHE1, which we hypothesized is important in activity S. pombe. Figure 1A is an alignment of the putative TM XI region of SpNHE1 with several other members of the Na + /H + exchanger family of proteins. Supplementary Fig. 1 illustrates the entire alignment of the protein. Multiple members of the family were included. Boxed and red amino acids highlight regions of conservation. There was little conservation beyond amino acid 383 of SpNHE1. This was a general trend in the protein, with regions intervening between transmembrane segments showing little conservation except in some more closely related species ( Supplementary Fig. 1). Figure 1B illustrates the putative structure of SpNHE1, which is based on molecular modeling of the protein described earlier 15 . A helical structure was predicted for residues 360-368 and residues 374-385, while an extended region was predicted between these two helical segments (Fig. 1B,C). According to the previous model of SpNHE1, Leu 386 to Ser 403 form an extended extracellular loop linking to putative TMXII. There was little conservation of this region of SpNHE1 with other family members.

NMR Resonance Assignment and Analysis. Well-resolved homonuclear 1 H-1 H TOCSY and NOESY
experiments and a natural abundance 1 H-13 C HSQC experiment were acquired in DPC micelles for a synthetic TM XI peptide. On the basis of these data, we were able to unambiguously sequentially assign all residues (chemical shifts are deposited in the BMRB, accession # 27092) Beyond the exhibition of appropriate spin-system properties for a given amino acid type, these assignments were developed on the basis of unambiguous sequential (i to i+1) and medium range (i to i+2 through i+4) NOE contacts ( Supplementary Fig. 2).
Through consideration of NOE contacts characteristic of helical structuring 16 alongside the observed Δδ values of H α , C α , and C β nuclei 17-19 calculated relative to random coil chemical shifts in a low dielectric environment 20 , a helical segment is clear over residues 18 Ala-Lys 21 (Fig. 2, corresponding to amino acids 375-386 of NHE1). In the N-terminal portion of the TM XI peptide, 1 Lys to 8 Val (containing amino acids 360-365) showed some degree of helicity according to Δδ-based predictions (Fig. 2), but this was not continuous. Similarly, DANGLE, which predicts secondary structuring based upon comparison of Δδ relative to random coil values in 8 M urea 22 to database inferences for a given sequence and structuring, predicted a long helical stretch in the C-terminal half of the TM IX segment and a short helical stretch in the N-terminal portion (Fig. 2). Unambiguous canonical helical NOE contacts were not observed in the N-terminal region, as chemical shift overlap was more problematic within this portion of the peptide.

Mutagenesis.
We made two sets of mutants in the putative TM XI to analyze the importance of amino acids in this region. The first set was an alanine scan of residues 360 to 393. The second set involved particular mutations of specific residues of this region, plus amino acid Ser 420 , which we hypothesized was associated with TM XI (Supplementary Table 1, Table 1). Initial experiments used western blot analysis to determine if the wild type and mutant SpNHE1 proteins were produced. Figure 3A-F demonstrates the results. All of the mutant proteins were expressed in S. pombe in amounts similar to that of the wild type. There was some small varying amounts of degradation of the SpNHE1 protein; however, the principal form of the protein was approximately 65 kDa in size. It was expressed at similar levels to the wild type SpNHE1 in all the mutants. The knockout strain that was not transfected with SpNHE1 showed no immunoreactivity. We also examined the localization of the wild type and mutant SpNHE1 proteins with defects in activity (see below). The wild type protein shared the same localization ( Fig. 3G) we have reported 23,24 . It was present on the plasma membrane and also at an intracellular location that may be perinuclear. The mutant SpNHE1 proteins had the same apparent localization as the wild type protein.
We (Supplementary Figure 3) then examined the ability of the wild type or mutant SpNHE1 protein to restore growth in salt containing medium. Supplementary Figure 3A-D illustrate the growth curves in NaCl containing medium. Wild type SpNHE1 protein restored growth to the knockout strain. Cells were able to grow robustly in solution containing 500 mM NaCl. In contrast, the knockout strain showed no growth in 500 mM NaCl and compromised growth in 200 mM NaCl. Mutation of several amino acids caused impairment of growth in high concentrations of NaCl. This was most notable with the K360A mutation (Supplementary 3A), which was almost comparable to the knockout strain. Mutations H367A and K383A also caused growth to be largely compromised in 500 mM NaCl. Several other mutants had more minor effects on the ability to tolerate higher concentrations of NaCl including E361A (summarized in Table 1). In the second round of mutagenesis, with mutations to other Scientific RepoRts | 7: 12793 | DOI:10.1038/s41598-017-12701-z amino acids, the proteins with mutations K360R, E361D and H367W conveyed NaCl resistance at levels equivalent or similar to that of the wild type protein. There was also a minor effect with K360E. The mutant proteins I371R, K383R and S420A did not convey sodium tolerance (Supplementary Figure 3E).
We also examined the same set of mutants for their ability to grow on solid media supplemented with NaCl. Figure 4A,B illustrates the results of growth on solid media with NaCl for the wild type and mutants, and the results are summarized in Table 1. The pattern of effects of the mutations on the ability to confer salt tolerance was largely the same as in liquid media. Mutant K360A and K383A proteins were unable to convey NaCl tolerance. Additionally, the H367A and L386A mutant proteins were largely ineffective in conferring NaCl tolerance. More minor effects were shown by the F364A, I371A, G372A, Y377A, M378A, F380A, L384A, and I391A (Table 1). Proteins with the mutation K360E, I371R, K383R and S420A were also defective in conveying salt tolerance on solid media, similar to the effect in liquid media.
The sensitivity of the mutants to LiCl was also examined in liquid and solid media (Supplementary Figure 4, Fig. 4C,D). Many mutants, with alanine mutations that were sensitive to NaCl, were also sensitive in similar degrees to LiCl. This was the case for F364A, H367A, Y377A, K383A and L386A. However, some mutants showed acute differences in their ion sensitivity. This was most notable for the K360A mutant strain, which lost virtually all NaCl tolerance, but was able to grow relatively well in the presence of external LiCl. The adjacent amino acid E361A had sensitivity to both LiCl and to NaCl. The cells with the mutant protein containing the amino acids I371A, G372A, M378A, and L381A were all more sensitive to LiCl than NaCl. Of the second group of mutations, proteins with the I371R, K383R and S420A did not confer resistance to LiCl, similar to what occurred with NaCl. The K360E mutant protein, conferred reasonable resistance to LiCl, in contrast to its weaker ability to do the same for NaCl (Summarized in Table 1).

Discussion
Na + /H + exchangers were earlier reported to have a unique fold that is hypothesized to be critical in transport by these proteins and is believed to part of the ion translocation core. The key features that have been noted are in the critical TM segments IV and XI. Both segments have been shown to be discontinuous helices that cross over each other in anti-parallel fashion with their crossing point within the lipid bilayer. Other amino acids neutralize the dipoles of the helices within the bilayer 11,14 . This arrangement was initially reported in NhaA of E. coli 11 , and has subsequently been shown in Thermos thermophilus (NapA) 12 , in the archaeal Na + /H + exchanger of Methanococcus MjNhaP1 25 and in Pyrococcus abyssi (PaNhaP) 13 . In PaNhaP and NapA, this structure is formed by helices 5 and 12 of a 13 TM segment protein, in contrast to the 12 TM segment protein NhaA.
While significant progress has been made in these species, in others there is not as much known about other NHE's. Notably, whether the same mechanisms of transport and protein conformation are maintained across species is not known. The S. pombe protein SpNHE1 is thought to be electroneutral in contrast to NhaA, which would certainly require some structural and functional differences 8,11 . From the sequence alignment, we noted that this yeast plasma membrane NHE also has some unique substitutions in TM XI. For example, Pro 370 is exclusively present in TMXI of SpNHE1 and yeast. Additionally, a conserved histidine (His 367 ) in TMXI segment is Table 1. Summary of growth of yeast strains containing wild type, or mutant SpNHE1 in liquid (L) or solid (S) media containing NaCl or LiCl. Mutations to amino acids are indicated. The measured or modeled propensity of an amino acid to be in a helix or extended conformation is indicated. * Cells with increased relative sensitivity to NaCl than to LiCl; **cells with increased relative sensitivity to LiCl than to NaCl; ***cells with sensitivity to both LiCl and NaCl; #cells with minor changes in sensitivity to LiCl and/or NaCl; h, NMR results suggest possible helicity; H, in a helical conformation; E, predicted or shown to be in an extended conformation. −, No growth; +,++,+++, increasing amount of growth with +++, indicating growth equivalent to wild type. −, not applicable or not measured.
unique to the yeast plasma membrane NHEs. Also, a hydrophobic amino acid (Ile 371 ) is present in yeast plasma membrane NHE1, which is Arg in other NHE1's. SpNHE1 may therefore have a novel or altered mechanism of Na + transport. SpNHE1 works to bring Na + out of the cell 9 in contrast to mammalian NHE1. These conserved unique substitutions may play a key role in function. We therefore examined the role of putative transmembrane segment XI in SpNHE1 which we hypothesized was critical in function. In this species, this protein is the key sodium tolerance protein. Its deletion causes salt sensitivity that makes it ideal to assay SpNHE1 function in live cells. We have earlier studied amino acids of this protein and defined several residues critical or important for function 9,15 . The 27 amino acids 360 KEALFVGHFGPIGVCAVYMAFLAKLLL 386 comprise a hydrophobic segment that was chosen for study. There were varied degrees of similarity of this segment to Na + /H + exchangers of other species (Fig. 1A), with other yeast species showing the greatest degree of similarity. In Thermos thermophilus (NapA), this segment of SpNHE1 aligned with TM 12, which is thought to be part of the ion translocation core. This was also the case with TM XI of E. coli NhaA, though in neither case was the similarity very high. In the case of MjNhaP1 and PaNhaP, the alignment with the corresponding regions of the ion translocation core is stronger.
In an earlier study we generated a SpNHE1 homology model (Fig. 1B,C) based on the E. coli NhaA structure and a comparative sequence alignment 15 . This model was validated using mutagenesis and structural analysis of TMIV 15 . Based on that model, a helical prediction of TMXI segment was made for residues 360-368 and residues 374-385, with an intervening non-helical segment (Fig. 1B, Table 1). To examine the structure of this transmembrane segment directly, we characterized a 27 amino acid peptide corresponding to residues 360-386 of SpNHE1 using solution-state NMR spectroscopy. It has been previously demonstrated that isolated peptides of TM segments contain most of the required structural information needed to form their native structures in membranes. For example, the solution structure of isolated segments of bacteriorhodopsin corresponded very well to the crystal structure of the protein [26][27][28] . Our results suggest that amino acids Lys 360 to Val 365 exhibited chemical shifts predominantly consistent with helicity, while a continuous helical segment was strongly indicated over residues Ala 375 -Ile 391 . These results are summarized in Table 1. This was essentially in agreement with the earlier modeling of the protein 15 , and is consistent with a break in helical character around the central proline residue of the segment 29,30 . A proline in the midst of a transmembrane segment of Na + /H + exchangers has earlier been suggested to cause an extended region within the lipid bilayer 31 . Overall, these results suggest that this TM segment retains the basic structure of the ion translocation domain. That is, a helix-extended region-helix motif. While the helical propensity of the N-terminal region of the segment was less pronounced, it was nonetheless present. It may be that interactions with hydrophobic regions of other transmembrane segments or lipids could stabilize a slightly more helical conformation 32 . We next asked the question, what is the functional importance of the individual amino acids of this transmembrane segment? Alanine scanning mutagenesis was used. Alanine has a small side chain that can substitute for most amino acids without disrupting the protein and at the same time alter the side chain. This kind of scan has been used earlier on SpNHE1 15 and other proteins 21,33 . All of the amino acids of this TM segment were mutated to alanine, aside from those that were already present as alanine. We then examined the ability of the expressed protein to rescue salt tolerance in the yeast knockout strain. Mutation to alanine (or other amino acids) did not affect the level of SpNHE1 protein expression. This result is consistent with what we observed earlier when amino acids of TM IV were mutated 15 . In contrast, when the mammalian NHE1 protein is mutated, many mutations affect both expression levels and targeting 34 . It appears as though that, at least for this class of proteins, in yeast, expression and targeting of the protein is more robust than for the mammalian NHE1 protein. We consider the effects of the amino acid substitutions in the light of the SpNHE1 model (Fig. 1B,C) and some of the amino acid interactions predicted in this model. It should be noted, that the model has yet to be definitely proven, nevertheless, some insights may be gained by this analysis. Nine of the mutations to alanine had more major effects on conferring resistance to Li, Na or both cations. Beginning from the N-terminus, there were two amino acids, Lys 360 and Glu 361 , that, when mutated, affected Na and Li tolerance. According to the previously hypothesized model, these two amino acids could be on or near the intracellular face of the membrane 15 . Mutation of these residues affecting ion specificity could be because they are involved in coordination of the internal cation, especially for negatively charged glutamic acid. Figure 5A illustrates their position on the surface of the protein. Replacement of the acidic Glu 361 with aspartic acid largely restored activity, supporting this hypothesis. It is also notable that this amino acid is largely conserved across many species (Fig. 1), though in some species it is replaced by an aspartate residue. Notably though, T. thermophilus NapA and E. coli NhaA do not contain an acidic residue at this location. A predicted topological assignment of this residue is at the cytoplasmic side and is located in close proximity to Glu 165 and Asp 355 . Asp 355 is conserved among the yeast species while Glu 165 is not conserved at all. Therefore, it is more likely that Glu 361 is directly involved in proton transport together with Asp 355 among the yeast group.
The side chain of the positively charged Lys 360 is predicted to point away from the central cavity (Fig. 5A) 15 . Its mutation affected ion specificity and this may be through an affect on the coordination sphere indirectly through alterations with other amino acids, that affect the sphere structure. Replacement of Lys 360 with a positively charged arginine largely restored function. Replacement of Lys 360 with a negatively charged glutamic acid had an intermediate effect, demonstrating that the charge requirement is not absolute ( Table 1). This amino acid is largely conserved across species (Fig. 1), though often replaced with arginine. A basic amino acid is present in NhaP1, NhaP, and NapA, though NhaA of E. coli is different, which may reflect specific differences in function and activity of this protein.
The next critical residue in SpNHE1 function was amino acid His 367 . Mutation of this residue to Ala severely impaired the ability of the protein to rescue salt tolerance, confirming an earlier result 9 . We have previously also shown that the H367R mutant is unable to transport Na, whereas the H367D mutant was also defective shifting the pH optimum to a more alkaline range. Analyzing the predicted SpNHE1 structure 15 suggests that the imidazole ring can make a hydrogen bonding interaction with hydroxyl group of Ser 420 (or Thr), which is present in TM XII of SpNHE1 (Fig. 5B). We therefore mutated Ser 420 to an alanine residue and examined the effect on protein function. The results demonstrated that this mutant protein is defective (Table 1). This supports the suggestion that there is an association that affects function. We also noted that in plant SOS1 protein, the equivalent position of His 367 is replaced with a tryptophan. Therefore, we examined if the H367W mutant can retain the protein's activity. We found that the H367W containing cells are not sensitive to Na and Li, supporting the idea that that the charge distribution on the imidazole nitrogen of histidine is responsible for the activity. Tryptophan has a nitrogen-containing ring in its indole side chain, which may perform a similar role to the imidazole side chain of histidine. Interestingly this residue is located in the discontinuous helix region.
More towards the center of the TM segment, both the I371A and G372A mutants had similar effects on salt tolerance, causing defects in both Na and Li tolerance, with a more pronounced effect on Li tolerance. Both of these residues are within the extended region of the TM segment (Fig. 2). Ile 371 is present in yeast plasma membrane NHEs and E.coli NhaA and is within the sequence GPIG. Sequence alignment (Fig. 1) identified the corresponding sequence motif in human NHE1, with an Ile to Arg substitution as 455 GGLRG 459 . The GLRG sequence is also observed in some plant antiporters. At the same time, in some other antiporters (MjNhaP1, PaNhaP, and TthNapA) the corresponding sequence was GPRG. The NMR structure of a peptide of this region of human NHE1 suggested that this part of the protein is in an extended conformation and is not helical 35 . Here, in the absence of a proline comparable to Pro 370 of SpNHE1, the two glycines (Gly 445 and Gly 446 ) may maintain flexibility.
Examination of the SpNHE1 model 15 suggests that Ile 371 may have hydrophobic interactions with some surrounding residues like Leu 148 , Phe 415 , and Leu 418 (Fig. 5C). The corresponding isoleucine in EcNhaA is Ile337 and is engaged in hydrophobic interactions 11 . Mutation of Ile 371 to Arg severely compromised activity of the protein (Table 1), supporting the contention that hydrophobic interactions are important in this location.
The G372A mutation had an effect similar to that of I371A, causing defective Na and Li tolerance. The exact cause of the effect is uncertain though the change in specificity does suggest an effect on ion coordination. A change from Gly to Ala might increase helical character, which could affect the extended conformation of this region. Glycine tends to allow flexibility, possibly acting as a hinge point-flexibility, and is rarely in a helix [36][37][38] . It may be that mutation to Ala interferes with the flexibility that Gly may induce in this region and that this affects ion coordination.
A segment of amino acids from Val 377 to Leu 384 , when mutated, all had either minor or major effects on the ability of SpNHE1 to confer salt tolerance. This is thought to be either a partially extended and helical region or a helical region ( Table 1). The effect of mutations Y377A, F380A, L384A (and nearby L386A) were minor. The mutants M378A, L381A, L384A and L386A all yielded varying degrees of a defective SpNHE1 protein. Modeling 15 of SpNHE1, suggests that these residues are in close proximity to a cluster of conserved hydrophobic amino acids. This includes the C-terminal portion of TM II (residues include Ile 78 , Val 79 , Leu 80 , and Val 82 ) and the C-terminal portion of TM IX (residues include Phe 305 , Phe 306 , Tyr 309 ) (Fig. 5D). The effect of these mutations may be due to reduced hydrophobic interactions between TM XI and these hydrophobic amino acids. It is interesting to note that in NhaA the equivalent helices are involved in helix-helix interactions as part of the scaffolding dimerization domain of NhaA. The recent structure of TtNapA also suggests that the C-terminal halves of TMIV and TMXI mediate scaffolding interactions with the dimerization domain 39 . The mutants M378A, L381A, L384A and L386 are all localized to the C-terminal half of TM XI of SpNHE1. If this region of the TM segment serves a similar role in SpNHE1 to that of the C-terminal of TM XI of TtNapA, this may explain their importance in function. They could be affecting dimerization of SpNHE1 through hydrophobic interactions with other amino acids. We have not demonstrated dimerization of SpNHE1, though this has been shown in the related protein AtSOS1 40 . SOS1 dimerization facilitates Na + transport 40 . Similar hydrophobic interactions through the C-terminal half of TM-XI of SpNHE1 could affect Na + transport through effects on dimerization.
The K383A mutation had a dramatic effect on activity of the protein and the K383R substitution only partially recovered activity. The larger size of this side chain may have affected interaction with other amino acids. This amino acid is conserved across the yeast and plant antiporters but not in some other species. Examination of the position of Lys 383 in the model of SpNHE1 15 suggests that this residue may interact with Asp 241 (Fig. 5E). We have previously shown that mutation of Asp 241 to Asn interferes with SpNHE1 function 9 . This supports the suggestion that there is an interaction between these amino acids.
Amino acid residues 387 to 393 showed no large effects with mutation to alanine. We earlier 10 showed that the mutations E390Q and D389N did not affect salt tolerance. This region seems relatively insensitive to mutation.
Overall, our results in this study can be compared with the human NHE1 protein. In SpNHE1, we found that the non-helical discontinuous part of TM XI spanned about 10 amino acid residues. A similar study of a peptide of TM XI of human NHE1 35 suggested that a comparable discontinuous region of TM IX was about 5 amino acids. Landau et al. 41 suggested a model of human NHE1 based on the crystal structure of E. coli NhaA. In this model amino acids 447-470 are proposed to be human TM XI and an even smaller discontinuous region is suggested in mid membrane centered around amino acid Leu 457 .
Another difference between human NHE1 and SpNHEI is that, SpNHE1 TM-XI does not contain an Arg (or Lys) at the center of the segment whereas human NHE1-TMXI contains Arg 458 there. The side chain of such a residue could be attracted to phospholipid head groups and could aid in conformational switching 42 . In the absence of an arginine, SpNhe1 TM-XI contains His 367 , which is located at the N terminus of the discontinuous region, and can attain a positive charge over certain pH ranges. The H367R mutant is incapable of transport confirming this is an important residue. However, as noted above, this residue might also interact with other amino acids such as Ser 420 . Three important residues L457, I461, and L465 in human NHE1-TMXI, have also been identified based on cysteine mutagenesis and are located either at the discontinuous region or at the C-terminal half of the TM 35 . An analogous effect of mutagenesis was observed for the C-terminal half of SpNhe1 where the mutations of the residues to alanine are moderately or highly affected protein activity.
In summary, based on NMR analysis, the deduced structure of a peptide of TMXI of SpNHE1 was that of a putative helix-extended region-helix. The functional data suggested that there were several more important regions on the transmembrane segment, where alteration to Ala compromised the protein's activity. When correlating effects of the mutations with molecular modeling of the protein, we came to the following putative roles of these amino acids on the protein. Glu 361 may be involved in cation coordination on the cytoplasmic face of the protein. His 367 , may be in association with Ser 420 and this is an important functional association. Ile 371 and Gly 372 are important in function. Ile 371 may have important hydrophobic interactions with other residues on transmembrane segments and Gly 372 may be important in maintaining an extended conformation of this region. A unique feature of SpNHE1 that we demonstrated, was that His 367 and Ile 371 are important functional residues of SpNhe1 and are conserved across related species. Residues from Val 377 to Leu 384 are important in function and may be important in hydrophobic interactions with other amino acids. Lys 383 of this region is also important in function and may be important in other ionic-electrostatic interactions with other amino acids. We suggest that TM XI of SpNHE1 has a similar structure and function to TM XI of E. coli, forming part of the ion translocation pathway that is characteristic of Na + /H + exchangers. It is possible that some mutations affect SpNHE1 indirectly, disrupting protein structure. Interpretations such as Glu 361 acting through an effect on cation coordination are based on molecular modeling, which remains to be confirmed by determination of the three dimensional structure of the protein.
Strains and media. S. pombe with the NHE1 gene disrupted (sod2::ura4) was used as a host to reintroduce wild type and mutant SpNHE1 protein 9 . The sod2::ura4 strain was maintained on low sodium minimal KMA medium or yeast extract adenine (YEA) as we described earlier 8,9 . KMA medium contains potassium hydrogen phthalate, 3 g; K 2 HPO 4 , 3 g; yeast nitrogen base without amino acids, 7 g; glucose, 20 g; and adenine, 200 mg (per 1 liter). Leucine at 200 mg/l was added to maintain the sod2::ura4 leu1-32 strain wherever indicated and all media was buffered using 50 mM MES/Citrate and pH adjusted to 5.0 with sodium free KOH. Wherever indicted NaCl or LiCl was added to the media at the indicated concentrations. The plasmid pREP-41sod2GFP was used to express the SpNHE1 protein as described earlier 23 . pREP-41sod2GFP contains the full length SpNHE1 gene with a C-terminal GFP tag separated by a nine amino acid Gly-Ala spacer. The GFP protein contains the Ser65Thr mutation and an NdeI site was removed by silent mutation to assist in cloning.
Transformation of the plasmid (and mutant forms) was into the sod2::ura 4 strain by electroporation 43 . Briefly, cells were grown in 100 ml of KMA (with Leucine) media at 30 °C with vigorous shaking until the OD600 reaches to 0.5 to 1.2. Cells were then incubated in ice for 15 min and harvested by centrifuging at 3500 × g for 5 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 200 ml of ice cold water. Washed cells were collected by centrifugation as above and further washed with 50 ml of ice-cold 1.0 M sorbitol twice. Finally, the cell pellet was resuspended in 1 ml of 1.0 M sorbitol. Resuspended cells were divided into 200 μL of aliquots and mixed with 0.1 ng of purified DNA. The cell-DNA mixture was transferred to a 0.2 cm electroporation cuvette pre-incubated in ice. After 5 min incubation an electric pulse was applied according to the Gene Pulser II (Bio-Rad) specifications. Immediately after pulsing, cells were resuspended in 800 μL of 1.0 M sorbitol and incubated at 30 °C without shaking for 1 hr. Cells were then centrifuged and spread in KMA agar containing 1.0 M sorbitol without leucine for growth and selection. Colonies appeared after 3-4 days.
Growth of transformed strains was in liquid and solid media. For growth curves in liquid media 5 × 10 6 cells were taken from an overnight exponentially growing culture. This was inoculated into 2.5 ml of fresh liquid media. S. pombe containing the pREP-41sod2GFP plasmid and mutants were routinely grown in medium in the absence of thiamine. Cultures were grown at 30 °C in a rotary shaker with constant agitation. The A 600 was determined at the indicated times. Growth curves were determined a minimum of three times and results are the mean + /− SE.
Growth on plates was examined in agar with KMA medium containing leucine supplemented with either NaCl or LiCl at the indicated concentrations. The pREP-41sod2GFP plasmid without mutations 23 was used as a control.