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PHYSIOLOGY AND BIOCHEMISTRY

The MrpA, MrpB and MrpD subunits of the Mrp antiporter complex in Bacillus subtilis contain membrane-embedded and essential acidic residues

Yusuke Kajiyama, Masato Otagiri, Junichi Sekiguchi, Toshiaki Kudo, Saori Kosono

  • 1Environmental Molecular Biology Laboratory, RIKEN, Japan
  • 2Department of Applied Biology, Shinshu University, Japan
  • 3Graduate School of Integrated Science, Yokohama City University, Japan
  • Correspondence
    Saori Kosono
    kosono{at}riken.jp

    • Microbiology 2009; 155(7):2137–2147 · https://doi.org/10.1099/mic.0.025205-0

    View at publisher PubMed

    Abstract

    Bacillus subtilis Mrp is a unique Na+/H+ antiporter with a multicomponent structure consisting of the mrpABCDEFG gene products. We have previously reported that the conserved and putative membrane-embedded Glu-113, Glu-657, Asp-743 and Glu-747 of MrpA (ShaA) are essential for the transport function. In this study, we further investigated the functional involvement of the equivalent conserved acidic residues of other Mrp proteins in heterologous Escherichia coli and natural B. subtilis backgrounds. Asp-121 of MrpB and Glu-137 of MrpD were additionally identified to be essential for the transport function in both systems. Glu-137 of MrpD and Glu-113 of MrpA were found to be conserved in the homologous MrpD/MrpA proteins as well as in the homologous subunits of H+-translocating primary active transporters such as Nuo and Mbh, suggesting their critical role in ion binding. The remaining essential acidic residues clustered in the C-terminal domain of MrpA (Glu-657, Asp-743 and Glu-747) and MrpB (Asp-121); these subunits are fused in some Gram-negative species. It is possible that the MrpA, MrpB and MrpD subunits, which contain essential transmembrane acidic residues, form the ion translocation site(s) of the Mrp antiporter complex.

    • A supplementary figure, showing the Na+ efflux curves using right-side-out membrane vesicles of B. subtilis cells, is available with the online version of this paper.

    Edited by: G. H. Thomas

    INTRODUCTION

    Mrp antiporters [reviewed by Swartz et al. (2005a); also designated Sha (Kosono et al., 2000), Pha (Putnoky et al., 1998), Mnh (Hiramatsu et al., 1998) and Sno (Bayer et al., 2006)] belong to the secondary cation–proton antiporter-3 (CPA-3) family (TC 2.A.63) in the transporter classification (TC) system (Saier, 2000). This family exhibits a particular multigene structure consisting of either seven or six members, while all the other monovalent CPAs are encoded by a single gene. A few mrp gene products characteristically show significant sequence similarity to the hydrophobic subunits of proton (H+)-translocating primary active transporters such as NADH–quinone oxidoreductases (NDH-1; Nuo for Escherichia coli NDH-1) and membrane-bound hydrogenases (Mbh) (Friedrich & Scheide, 2000; Mathiesen & Haegerhaell, 2003). Although Mrp antiporters are apparently similar to oxidoreduction-driven transporters with regard to the features of the multisubunit structures and protein sequences, it has not yet been clearly shown that they have a primary mode of transport (Hiramatsu et al., 1998; Ito et al., 2001; Swartz et al., 2005b). Mrp antiporters are widely distributed in bacteria, including pathogens, and archaea that inhabit various environments; however, they are apparently not found in members of the family Enterobacteriaceae, such as E. coli. Mrp antiporters not only play a critical role in the homeostasis of monovalent cations but also have been shown to be involved in sporulation (Kosono et al., 2000), virulence (Kosono et al., 2005), symbiotic nitrogen fixation (Putnoky et al., 1998), susceptibility to microbicidal proteins (Bayer et al., 2006), arsenite oxidation (Kashyap et al., 2006) and photosynthesis (Blanco-Rivero et al., 2005). It is intriguing to know how Mrp antiporters are involved in such diverse physiological functions that are apparently unrelated to each other.

    It has recently been demonstrated that the Mrp antiporter in Bacillus subtilis (Kajiyama et al., 2007) and Bacillus pseudofirmus (Morino et al., 2008) is actually a multicomponent antiporter consisting of the mrpABCDEFG gene products. However, the role played by each subunit in the transport function of the antiporter remains to be elucidated. Furthermore, the subunit(s) that forms the ion translocation site(s) remains to be identified. Of the mrp gene products, MrpA, MrpC and MrpD show sequence similarity to membrane-embedded NuoL, NuoK, and NuoM as well as NuoN, of the H+-translocating NDH-1 of E. coli, respectively. MrpA and MrpD are homologous to each other and also show sequence similarity to MbhH (or Mbh8), which is a hydrophobic subunit of the H+-translocating Mbh of Pyrococcus species (Friedrich & Scheide, 2000; Mathiesen & Haegerhaell, 2003). This implies that these subunits are involved in cation binding and translocation. In many cation transporters, the essential acidic residues in transmembrane helices are reported to be involved in cation binding and/or translocation (Hunte et al., 2005; Inoue et al., 1995; Meier et al., 2005; Murata et al., 2005; Murtazina et al., 2001; Takase et al., 1999). We have previously reported the functional involvement of the conserved transmembrane acidic residues of B. subtilis MrpA (ShaA) by using heterologous E. coli as the host, and identified that Glu-113, Glu-657, Asp-743 and Glu-747 are essential for the transport function (Kosono et al., 2006). Such conserved transmembrane acidic residues are also found in other Mrp proteins. To understand the molecular architecture of the structurally and functionally unique Mrp antiporters, especially to identify the subunit(s) that forms the ion translocation site(s), it is necessary to identify the essential residues in all the Mrp proteins. Here, we studied the functional involvement of the conserved transmembrane acidic residues (Glu or Asp) in other Mrp proteins, besides MrpA, that may be involved in sodium ion (Na+) and/or H+ translocation, in the heterologous E. coli and natural B. subtilis backgrounds.

    METHODS

    Determination of multiple alignments and prediction of transmembrane topology.

    Multiple alignments were determined by clustal_x (Thompson et al., 1997) and transmembrane topology was predicted by six methods (SOSUI, TMHMM, HMMTOP, TMpred, PHDhtm and DAS), as described previously (Kosono et al., 2006). The HMMTOP method was also used to predict the orientation of proteins in the membrane.

    Construction of B. subtilis mrp-expressing plasmids.

    The pTY11 plasmid expressing the intact mrp cluster under the control of the lacZ promoter was constructed previously (Kosono et al., 2006). Site-directed mutagenesis was performed using a PCR-based technique. The mrp (sha)-back fragment in the pTY11 plasmid was replaced with the corresponding fragments containing a site-directed mutation in mrpB, mrpD, mrpE and mrpF, as described previously (Kosono et al., 2006). The resulting plasmids are listed in Table 1⇓. They were transformed into the major Na+/H+ antiporter-deficient E. coli KNabc (ΔnhaA : : Kmr, ΔnhaB : : Emr, ΔchaA : : Cmr) (Nozaki et al., 1996) in order to examine the ability to complement the NaCl-sensitive phenotype and balance the antiporter-deficient antiport activity.

    Table 1.

    Effect of site-directed mutations on the activity of the Bs-Mrp antiporter examined in E. coli Knabc

    WT, wild-type; NC, negative control.

    Construction of B. subtilis strains.

    B. subtilis strains (SK702, KY8, KY10, KY11 and KY12), in which MrpA, MrpB, MrpD, MrpE and MrpF are functionally replaced by their corresponding histidine (His)-tagged forms, were constructed as described previously (Kajiyama et al., 2007); these strains are derived from B. subtilis UOT1285 (trpC2 lys-1 aprEΔ3 nprE18 nprR2) (Yamashita et al., 1986). Site-directed mutagenesis was performed using a PCR-based technique, as described previously (Kosono et al., 2006). PCR-amplified mrp gene fragments containing a site-directed mutation, a ribosome-binding site (5′-AAAGGAGGA-3′) at the 5′ end, and a His-tag-coding sequence (5′-CATCACCATCACCATCAC-3′) at the 3′ end were cloned into the SalI/EcoRI sites of pAPNC213 (Morimoto et al., 2002). The resulting plasmids were transformed into each corresponding mrp-deleted mutant, as described previously (Kajiyama et al., 2007). The genes encoding the mutated Mrp proteins were integrated into the aprE site by homologous recombination and expressed under the control of the IPTG-inducible Pspac promoter from pAPNC213. The resulting strains are listed in Table 2⇓.

    Table 2.

    Effect of site-directed mutations on the activity of the Bs-Mrp antiporter examined in B. subtilis

    WT, wild-type; NC, negative control.

    Growth experiments.

    Assays of the NaCl resistance of E. coli KNabc were performed as described previously (Kosono et al., 2006). For assays of the NaCl resistance of B. subtilis, fresh colonies grown on minimal medium with potassium (MMK) plates (Yoshinaka et al., 2003) containing 50 μg spectinomycin ml−1 were collected and suspended in NaCl-free Luria–Bertani (LB)-Tris medium [10 g tryptone l−1, 5 g yeast extract l−1, and 10 mM Tris/HCl (pH 7.0)]. The cell suspensions were inoculated into LB-Tris medium containing 1 mM IPTG and the indicated concentrations of NaCl to adjust the OD660 to 0.05. Growth was evaluated by OD660 after 8 h of cultivation at 37 °C.

    Transport assay.

    The Na+/H+ antiport activity was measured by a quenching method, as described previously (Kosono et al., 2006). The antiport activity was calculated by subtracting the percentage of dequenching (relative to quenching on addition of 2 mM lactate) without addition of NaCl from that on addition of 10 mM NaCl.

    Assays of 22Na+ efflux energized by an inward pH gradient were performed as described previously (Kosono et al., 1999). Right-side-out vesicles were prepared from B. subtilis cells in acetate buffer (pH 6.0; 100 mM potassium acetate, 5 mM MgCl2, 10 mM sodium phosphate). Downhill 22Na+ efflux was initiated by diluting the membrane vesicles (20 μg protein) in gluconate buffer (pH 6.0; 100 mM potassium gluconate, 5 mM MgCl2, 10 mM potassium phosphate) or acetate buffer. At the time points of 5, 10 and 15 s, the reaction was terminated by filtration through a 0.45 μm pore-size filter. The filter was washed and dried, and the radioactivity retained on the filter was counted by liquid scintillation spectrometry. Na+ efflux was calculated as the percentage of the decrease of the radioactivity in the vesicles relative to the total radioactivity loaded into the vesicles. The total radioactivity loaded into the vesicles was calculated by subtracting the radioactivity in the vesicles treated with gramicidin (the background activity) from that in the vesicles at time 0. The relative activity of Na+ efflux with respect to the corresponding wild-type was calculated as:

    Figure image not available in archive
    where [efflux]Mutant indicates efflux from the mutant, [efflux]NC indicates efflux from the negative control, and [efflux]WT indicates efflux from the wild-type.

    Western blotting.

    Membrane samples obtained from E. coli KNabc and B. subtilis cells were prepared as described previously (Kajiyama et al., 2007; Kosono et al., 2006). The samples were loaded onto an SDS-polyacrylamide gel without boiling. The proteins were electrophoretically transferred to a PVDF membrane by using a semidry transfer system. The transferred membranes were soaked for 1 h in Tris-buffered saline [TBS; 50 mM Tris/HCl (pH 7.5), 150 mM NaCl] containing 5 % (w/v) skimmed milk for blocking. A polyclonal anti-His-tag antibody (MBL) or anti-MrpE antibody (Kosono et al., 2006) diluted to 1 : 2000 with Can Get Signal solution (Toyobo) was used. Alkaline phosphatase-conjugated secondary antibodies were then used at 1 : 20 000 dilution. The signals were visualized using nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) solution (Roche).

    Pull-down assay.

    To examine the association of each His-tagged Mrp protein with MrpE, a pull-down assay was performed as described previously (Kajiyama et al., 2007). Membrane fractions were solubilized in TMKN buffer [30 mM Tris/HCl (pH 7.4), 5 mM MgCl2, 5 mM KCl, 500 mM NaCl] containing 1 % (w/v) n-dodecyl-β-d-maltoside (DDM) and 1 mM PMSF for 1 h at 4 °C. After centrifugation at 100 000 g for 1 h, the supernatant containing the solubilized membrane proteins was incubated with 0.5 ml bed volume of equilibrated TALON metal affinity resin (Clontech) for 1 h at 4 °C. Unbound proteins were washed away with 25 ml TMKN buffer containing 0.05 % DDM and 10 mM imidazole, and then eluted with 5 ml TMKN buffer containing 0.05 % DDM and 250 mM imidazole. The eluted sample was concentrated by ultrafiltration using an Amicon Ultra-4 filter device (30 000 molecular weight cut-off; Millipore). The concentrated sample containing 5 μg protein was subjected to SDS-PAGE and Western blotting with an anti-His-tag or anti-MrpE antibody.

    RESULTS

    Conserved transmembrane acidic residues of the B. subtilis MrpB, MrpD, MrpE and MrpF proteins

    We have previously reported the functional involvement of the conserved acidic residues in the predicted transmembrane helices of B. subtilis MrpA (ShaA) (Kosono et al., 2006). We further investigated such conserved transmembrane acidic residues in other Mrp proteins. A total of six candidate residues were found in MrpB, MrpD, MrpE and MrpF. MrpB was predicted to possess four transmembrane segments (TMs) and contained a well-conserved Asp-121 in TM-4 (Fig. 1a⇓). MrpD was predicted to have 14 TMs and contained conserved Asp-75 in TM-3, Asp-128 in TM-4 and Glu-137 in TM-5 (Fig. 1b⇓). MrpD and the N-terminal region (TM-1–TM-13) of MrpA consisted of similar TMs, and Asp-75, Asp-128 and Glu-137 of MrpD were positioned equivalently to Asp-50, Asp-103 and Glu-113 of MrpA, respectively. These residues were also conserved in NuoM and MbhH (Fig. 1b⇓). MrpE was predicted to possess four TMs and contained Glu-67; this residue was conserved in Gram-positive bacteria and was substituted with Asp in Gram-negative bacteria (Fig. 1c⇓). MrpF was predicted to possess three TMs and contained a well-conserved Asp-38 in TM-2 (Fig. 1d⇓). We considered that such conserved residues were candidates for involvement in Na+ and/or H+ binding. MrpC (Asp-66 and Asp-101) and MrpG (Asp-32) also contained conserved acidic residues, but in our prediction of the transmembrane topology, they were not located in the TMs.

    Figure image not available in archive
    Fig. 1.

    Alignment and transmembrane topology of MrpB (a), MrpD (b), MrpE (c) and MrpF (d) homologues from selected species. Multiple alignment was performed by clustal_x and prediction of transmembrane topology was performed by six methods (SOSUI, TMHMM, HMMTOP, TMPred, PHDhtm and DAS), as described previously (Kosono et al., 2006). Asterisks show amino acid residues conserved in all sequences. Exclamation marks show acidic residues conserved in homologues. The six acidic residues characterized in this study are shown in red type. The stretches of residues in which four or more of the six methods agreed in their prediction of a TM are highlighted in yellow and areas in which three or fewer methods agreed are highlighted in cyan. The predicted orientation of the TMs is indicated by i (inside) and o (outside), based on the analysis of B. subtilis Mrp by the HMMTOP method only. Bsu, B. subtilis; Sau, Staphylococcus aureus; Pae, Pseudomonas aeruginosa; Sme, Sinorhizobium meliloti; Pfu, Pyrococcus furiosus; Eco, E. coli.

    Evaluation of the effect of the site-directed mutations of conserved acidic residues in the heterologous E. coli background

    We first determined the functional involvement of the above mentioned six residues in ion transport by using heterologous E. coli as the host. The six conserved acidic residues were replaced by site-directed mutagenesis with alternative acidic residues (Asp with Glu and Glu with Asp), corresponding amide residues (Asp with Asn and Glu with Gln) or alanine. The pTY11 derivatives harbouring a site-directed mutation in the mrp cluster were introduced into E. coli KNabc, and the ability of the strain to complement the Na+-sensitive phenotype and balance the Na+/H+ antiport activity was examined (Table 1⇑). The D121A and D121N mutants of MrpB, the E137A and E137Q mutants of MrpD, and the D38A and D38E mutants of MrpF showed no ability to complement the Na+-sensitive phenotype of E. coli KNabc, suggesting that these residues were essential for the Mrp function. The antiport activity of the variants was negligible or low compared with that of the wild-type. All the mutants of Glu-67 of MrpE substantially retained the ability to restore growth and antiport activity. This result is consistent with that reported by Morino et al. (2008), who state that the G67A mutation of B. pseudofirmus (Bp)-MrpE, which is equivalent to Glu-67 of Bs-MrpE, shows no significant phenotype. All the mutants of Asp-75 of MrpD showed a partially impaired growth phenotype and low antiport activity. The D128E and D128N mutants of MrpD showed a growth phenotype similar to that of the wild-type but a low antiport activity.

    Because the expression of the mutated Mrp has not been determined directly in E. coli, the results obtained with this host do not have straightforward implications; the mutations may affect expression, assembly or functionality. We examined the level of MrpE protein in the membrane fractions to determine whether the mutations affected its expression (Fig. 2⇓). We detected similar levels of MrpE protein in the membrane fractions of the mutants, except in those of the D75A and D128A mutants of MrpD and the three D38 mutants, especially D38A, of MrpF. Therefore, we could not exclude the possibility that the impaired expression of MrpE caused the above phenotypes.

    Figure image not available in archive
    Fig. 2.

    Western blots to detect MrpE proteins in the membrane fractions of E. coli KNabc. Membrane samples (containing 15 μg protein) were prepared from KNabc containing the plasmids listed in Table 1 and loaded onto SDS-polyacrylamide (15 %) gels without boiling. MrpE protein (indicated by arrows) was probed with an anti-MrpE antibody. The membrane samples of pTWV228- and pTY11-transformed KNabc were used as the negative control (NC) and positive control (wild-type; WT), respectively. The experiment was repeated three times and representative data are shown.

    Evaluation of the effect of the site-directed mutation of conserved acidic residues in the natural B. subtilis background

    To further confirm the above results, we next examined the effect of site-directed mutations in the natural B. subtilis background on the basis of the complementation of mrp-deleted strains (Kajiyama et al., 2007). The mutated mrpB, mrpD, mrpE and mrpF genes with a His-tag-coding sequence were expressed under the control of the IPTG-inducible Pspac promoter at the aprE site of each corresponding mrp-deleted strain genome, and the effects of the site-directed mutations were evaluated from the extent of the complementation of the Na+-sensitive growth phenotype as compared with the corresponding wild-type Mrp. We constructed two types of variants of each acidic residue in which it was replaced by an alternative acidic residue or a corresponding amide residue.

    All mrp-deleted strains do not grow on LB agar plates containing 0.2 M NaCl, but growth in the presence of 1 M NaCl is restored by complementation with the His-tagged forms of each wild-type Mrp (Kajiyama et al., 2007). This was true under liquid culture conditions (Table 2⇑). As shown in Table 2⇑, KY105 (the D121N mutant of MrpB) showed no ability to grow in 0.2 M NaCl, and was equivalent to KY90 [the negative control (NC) strain], while KY104 (D121E) grew in 1 M NaCl to the same extent as KY8 [the wild-type (WT)-complemented strain]. This result was consistent with that obtained with the E. coli system (Table 1⇑), and it was considered that Asp-121 of MrpB was essential and its carboxyl group necessary as well as sufficient for the function. In MrpD, KY114 (E137Q) showed almost no ability to grow in 0.2 M NaCl, suggesting that Glu-137 is functionally important. KY113 (E137D) showed growth in 1 M NaCl (OD660 1.28), although this was less than that of the wild-type and other mutants (2.87–3.46). We did not find impaired phenotypes for the mutants of Asp-75 and Asp-128 with the B. subtilis system. In MrpE, KY116 (E67D) and KY117 (E67Q) showed no significant phenotypes, suggesting that Glu-67 is dispensable for the function; this was consistent with the results observed with E. coli (Table 1⇑). The result for Asp-38 of MrpF differed from that obtained with E. coli; both KY119 (D38E) and KY120 (D38N) showed substantial growth in 1 M NaCl (3.58 and 2.65, respectively), which was more extensive than that of KY12 (wild-type, 1.80) and similar to that of the other wild-type-complemented strains (2.87–3.71). Thus, the results suggested that Asp-38 is dispensable for this function in the B. subtilis system.

    We also confirmed the phenotypes of the site-directed mrpA mutants that were previously studied by using heterologous E. coli KNabc (Kosono et al., 2006). It was confirmed that Glu-113, Asp-743 and Glu-747 are essential for function from the severely impaired phenotypes of their mutants; the carboxyl group of Asp-743 is necessary as well as sufficient [compare SK712 (D743E) and SK713 (D743N)], while Glu-113 and Glu-747 are irreplaceable (Kosono et al., 2006). SK711 (E657Q) showed a small but definite ability to grow in 0.2 M NaCl (OD660 0.94), which was not observed in E. coli (Kosono et al., 2006). The partially impaired phenotypes of the Asp-103 variants were confirmed, but their complementation abilities were reversed in B. subtilis and E. coli (Kosono et al., 2006); SK706 (D103E) showed more extensive growth than SK707 (D103N). The Asp-50 variants showed contradictory phenotypes in a previous study with E. coli; examined by complementation of the growth phenotype, their ability was similar to that of the wild-type, but their antiport activity was significantly lower (Kosono et al., 2006). With regard to the complementation in B. subtilis, the Asp-50 variants showed partially impaired phenotypes and also Na+ efflux activity (Table 2⇑), suggesting that the residue has a role in the transport function.

    Na+ efflux activity from right-side-out membrane vesicles obtained from the B. subtilis strains

    We next examined the Na+ efflux activity from right-side-out membrane vesicles upon energization of a transmembrane proton gradient (ΔpH), a component of proton motive force (Table 2⇑, Supplementary Fig. S1). The Na+ efflux activity of KY89, KY90, KY91 and KY93 (ΔmrpA, ΔmrpB, ΔmrpD and ΔmrpF, respectively) was enhanced by 20–31 % by complementation with each wild-type mrp. KY92 [ΔmrpE (shaE)] retained higher Na+ efflux activity (56 %) than the other Δmrp strains (35–47 %); this result was consistent with our previous results (Yoshinaka et al., 2003). Morino et al. (2008) have also reported that the Mrp antiporter in B. pseudofirmus (Bp-Mrp) shows a residual activity in the absence of MrpE. The efflux activity of the mutants was evaluated as relative activity with respect to the corresponding wild-type, as shown in Table 2⇑. We detected efflux activities that were mostly consistent with the phenotypes in the growth test. In MrpA, SK709 (E113Q), SK713 (D743N) and SK715 (E747Q), with negligible growth in 0.2 M NaCl, showed negligible activity (0.04–0.2× wild-type levels). SK710 (E657D) and SK712 (D743E) showed activity equivalent to that of the wild-type (1.0–1.2× wild-type). In MrpB, KY104 (D121E) showed activity similar to that of the wild-type (0.8× wild-type), while the D121N mutant showed activity equivalent to that of the negative control (0× wild-type). In MrpD, the KY107 (D75E), KY108 (D75N), KY110 (D128E) and KY111 (D128N) mutants showed activity similar to that of the wild-type (0.81–1.0× wild-type), KY114 (E137Q) showed activity equivalent to that of the negative control (0× wild-type) and KY113 (E137D) showed intermediate activity (0.48× wild-type); this was consistent with the growth phenotypes. In MrpE, KY116 (E67D) and KY117 (E67Q), which showed growth similar to that of the wild-type in 1 M NaCl, showed efflux activity equivalent to that of the wild-type (1.0–2.0× wild-type), although the difference between the wild-type and the negative control was barely detectable. In MrpF, KY119 (D38E) and KY120 (D38N) also showed activity equivalent to that of the wild-type (0.80–1.1× wild-type).

    Our Na+ efflux data were only semiquantitive and not always consistent with growth ability; for example, SK707 (MrpA-D103N) and KY92 (ΔmrpE) showed efflux of 56 %, although the former grew in 0.6 M NaCl and the latter showed no growth in 0.2 NaCl (Table 2⇑). However, our efflux data strongly supported the evidence from the growth assays that Asp-121 of MrpB and Glu-137 of MrpD, in addition to the previously identified Glu-113, Glu-657, Asp-743 and Glu-747 of MrpA, were essential for Mrp function. The carboxyl group of Asp-121 of MrpB was necessary as well as sufficient for Mrp function, but this was not true in the case of Glu-137 of MrpD. On the other hand, Glu-67 of MrpE and Asp-38 of MrpF were likely dispensable for Mrp function.

    Confirmation of the expression and assembly of mutated Mrp proteins in the membrane by Western blotting and pull-down assay

    An advantage of using B. subtilis as the host is that the His-tag at the C-terminus of the mutated proteins is available for detection. We examined the levels of the mutated Mrp proteins in the membrane fractions by Western blotting with an anti-His-tag antibody. As shown in Fig. 3⇓, except for the D50E mutant of MrpA, which showed a lower level than that of the wild-type, equivalent or higher levels of the other mutant derivatives of MrpA, MrpB, MrpD, MrpE and MrpF were detected in comparison with their corresponding wild-type forms. Thus, we consider that the observed phenotypes are not likely to be due to the impaired expression of the mutated Mrp proteins. Of course, we cannot completely neglect the possibility that the overexpressed mutated proteins cause the impaired phenotypes. We detected a lower amount of the wild-type MrpF compared with the mutant variants, and this might explain the reduced growth of KY12 (wild-type) compared with KY119 (D38E) and KY120 (D38N) (Table 2⇑).

    Figure image not available in archive
    Fig. 3.

    Western blots to detect the wild-type and mutated Mrp proteins in membrane fractions of B. subtilis. Membrane samples were prepared from the B. subtilis strains listed in Table 2 and loaded onto SDS-polyacrylamide gels without boiling under the following conditions: 20 μg protein onto 12.5 % acrylamide (MrpA), 40 μg protein onto a 5–20 % acrylamide gradient (MrpB), 25 μg protein onto 15 % acrylamide (MrpD), 50 μg protein onto a 10–20 % acrylamide gradient (MrpE), and 25 μg protein onto 15 % acrylamide (MrpF). His-tagged Mrp proteins (indicated by arrows) were probed with an anti-His-tag antibody. The membrane sample of UOT1285 was used as the negative control (NC). For MrpA, WT and WT* indicate the original and synonymous variants, respectively (Kajiyama et al., 2007). The experiment was repeated three times and representative data are shown.

    The MrpA–MrpG proteins constitute a complex in the membrane (Kajiyama et al., 2007; Morino et al., 2008), and the site-directed mutations may affect the assembly of the Mrp complex. We confirmed the association of MrpE with the mutated MrpA, MrpB and MrpD that showed impaired phenotypes by pull-down analysis, as described previously (Kajiyama et al., 2007) (Fig. 4⇓). All the His-tagged MrpA, MrpB and MrpD mutants tested coeluted normally with MrpE, indicating that the association of the mutated Mrp variants with MrpE was not affected. Thus, we consider it likely that the site-directed mutations in this study did not affect the complex formation of the Mrp proteins.

    Figure image not available in archive
    Fig. 4.

    Pull-down assays using the membrane samples of the MrpA, MrpB and MrpD derivative strains shown in Table 2. Eluted samples containing 5 μg protein from TALON metal affinity resin were loaded onto 10–20 % acrylamide gradient gels without boiling. Immobilized wild-type and mutant MrpA, MrpB and MrpD (indicated by arrows) successfully pulled down MrpE (indicated by stars). The membrane sample of UOT1285 was used as the negative control (NC). A synonymous variant of MrpA (WT*) (see legend of Fig. 3) was used as the wild-type of MrpA. The experiment was repeated three times and representative data are shown.

    DISCUSSION

    In our present and previous studies (Kosono et al., 2006), we identified a total of six functionally important transmembrane acidic residues of the Mrp antiporter, the schematic positions of which are shown in Fig. 5⇓. The essential acidic residues were located in the MrpA, MrpB and MrpD subunits of the Mrp complex. Glu-113 of MrpA and Glu-137 of MrpD were located at the corresponding positions and were also conserved in NuoL, NuoM and MbhH (see Fig. 1b⇑). A recent report states the interesting finding that the corresponding Glu-144 of E. coli NuoM is essential for NDH-1 activity and H+ translocation (Torres-Bacete et al., 2007). In light of these facts, we strongly suggest that both the Glu residues of MrpA and MrpD play a critical role in the transport function and are probably involved in H+ and/or Na+ translocation. The remaining four essential residues, in most of which the carboxyl group is necessary as well as sufficient for the Mrp function, were concentrated in the C-terminal region of MrpA (Glu-657, Asp-743 and Glu-747) and MrpB (Asp-121); the corresponding peptides are fused in some Gram-negative species. Thus, these acidic residues might assemble close to each other in order to form a domain in the complex. It has been recently shown that B. pseudofirmus MrpA, MrpB, MrpC and MrpD form a stable subcomplex in the membrane (Morino et al., 2008). Taking all these findings into consideration, we suggest that the MrpA, MrpB and MrpD subunits, which contain the essential acidic residues, form a translocation pathway in the Mrp antiporter complex.

    Figure image not available in archive
    Fig. 5.

    Schematic positions of functionally important acidic residues of Mrp proteins identified in this study. Irreplaceable Glu-113 and Glu-747 of MrpA are indicated by stars, and the Glu-657 and Asp-743 of MrpA and also Asp121 of MrpB, in which the carboxyl group is necessary and sufficient, are indicated by dots. The Glu-137 of MrpD, which does not fall into the above categories, is indicated by a diamond. The region that is homologous between the N terminals of MrpA and MrpD is shown in grey. The MrpA and MrpB subunits are fused in some Gram-negative bacterial species (indicated by the dotted line).

    With regard to the MrpF mutants, we obtained contradictory results between the E. coli and B. subtilis systems; the mutations in MrpF significantly affected the function in E. coli, while they had no significant effect in B. subtilis. The MrpE levels in the membrane fraction of E. coli KNabc with the mrpF mutations were substantially lower than those in other strains (Fig. 2⇑), and this may have caused the impaired phenotype of the MrpF mutants. In contrast, we confirmed the expression of mutated MrpF proteins in the membrane and their association with MrpE in B. subtilis. Thus, we consider that the result obtained for B. subtilis is more reliable.

    In several transporters whose structures have been determined by X-ray crystallography, a structural symmetry is observed around the substrate-binding sites. In the secondary Na+/H+ antiporter NhaA of E. coli, the probable Na+/Li+-binding site (Asp-163 and Asp-164) is positioned near the centre of two structurally related bundles (Hunte et al., 2005). The E. coli lactose/H+ symporter LacY has a clear twofold symmetry between the N- and C-terminal domains, and the sugar-binding site is in the vicinity of the approximate molecular twofold axis of LacY (Abramson et al., 2003). The drug/H+ antiporter EmrE is a homodimeric transporter, in which two membrane-embedded Glu-14 residues at the dimerization interface bind drugs and protons in turn during translocation (Pornillos et al., 2005). These observations lead us to speculate that the homologous MrpA and MrpD subunits that contain an essential Glu residue constitute the core of the ion translocation site.

    Na+-binding sites have been defined in high-resolution crystal structures of Enterococcus hirae V-type Na+-ATPase (Murata et al., 2005), Ilyobacter tartaricus F-type Na+-ATPase (Meier et al., 2005) and LeuTAa, a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters (Yamashita et al., 2005). Four or five oxygen atoms derived from the polypeptide (and also the carboxyl oxygen of the substrate leucine for the Na1 binding site in LeuTAa) participate in sodium coordination. Of the residues that coordinate the Na+, Glu-139 of Enterococcus hirae V-ATPase and Glu-65 of I. tartaricus F-ATPase are essential and are highly conserved among the c subunits of both Na+- and H+-translocating V- and F-ATPases, suggesting that they are critical for Na+ or H+ binding (Meier et al., 2005; Murata et al., 2005). The replacement of Glu-139 of Enterococcus hirae V-ATPase by aspartate abolishes this activity (Takase et al., 1999). In contrast, for the essential Glu-137 of the yeast H+-translocating V-ATPase, which is the equivalent of Glu-139 of Enterococcus hirae, the activity of the polypeptide is retained even after its replacement by aspartate (Noumi et al., 1991). The H+-translocating F0F1-ATPase in E. coli contains the essential Asp-61 residue, which is equivalent to the Glu-65 residue that participates in Na+ binding in I. tartaricus F-ATPase (Meier et al., 2005); the Asp-61 to Glu mutant retains a partial activity of the polypeptide (Miller et al., 1990). The mean distances between the Na+ and the ligand oxygen atoms are 2.25 Å (0.225 nm) for Enterococcus hirae V-type Na+-ATPase (Murata et al., 2005), 2.37 Å (0.237 nm; σ=0.06) for I. tartaricus F-type Na+-ATPase (Meier et al., 2005) and 2.28 Å (0.228 nm; σ=0.15) for LeuTAa (Yamashita et al., 2005). We considered that the distance between the Na+ and the side-chain oxygen was critical for Na+ binding. Thus, an essential acidic residue at the binding site may be irreplaceable because the replacement of Glu by Asp or vice versa will result in a decrease or increase of 1.5 Å (0.15 nm), which is not negligible when compared with the dimensions of the original structures. In contrast, the carboxyl group, rather than the length of the side chain, is more important for proton binding, and thus the interchange between Glu and Asp is permissible in the cases of the H+-translocating counterparts. Consistent with our idea, in the case of Glu-144 of NuoM, which is strongly suggested to participate in H+ translocation, the E144A or E144Q mutation abolishes the NDH-1 activity and H+ translocation, but the E144D mutation has a negligible effect upon them (Torres-Bacete et al., 2007). From this point of view, Glu-113 and Glu-747 of MrpA, which could not be replaced by aspartate, could be involved in Na+ binding, while the remaining residues (Glu-657 and Asp-743 of MrpA, and Asp-121 of MrpB, with Glu-137 of MrpD, which is close to this group) might be involved in H+ binding. It is necessary to elucidate the crystal structure of the Mrp complex to precisely understand the arrangement of these essential residues in the complex. This study will provide experimental evidence to aid understanding of the molecular architecture of the Mrp complex.

    Acknowledgments

    We are grateful for the support provided by RIKEN Brain Science Institute (BSI)'s Research Resources Center for DNA sequencing analysis. This work was partially supported by grants for Bioarchitect Research, Eco-Molecular Research, and Discovery Research Institute (DRI) Research programs from RIKEN. This work was also supported by a Grant-in-Aid for Scientific Research (C) to S. K.

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