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Biochem. J. (2013) 454, 559–570 (Printed in Great Britain) 559 doi:10.1042/BJ20130388 Channel-mediated lactic acid transport: a novel function for aquaglyceroporins in bacteria Gerd P. BIENERT*1,2 , Benoı̂t DESGUIN*1 , François CHAUMONT*3 and Pascal HOLS*3 *Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 4-5, 1348 Louvain-la-Neuve, Belgium MIPs (major intrinsic proteins), also known as aquaporins, are membrane proteins that channel water and/or uncharged solutes across membranes in all kingdoms of life. Considering the enormous number of different bacteria on earth, functional information on bacterial MIPs is scarce. In the present study, six MIPs [glpF1 (glycerol facilitator 1)–glpF6] were identified in the genome of the Gram-positive lactic acid bacterium Lactobacillus plantarum. Heterologous expression in Xenopus laevis oocytes revealed that GlpF2, GlpF3 and GlpF4 each facilitated the transmembrane diffusion of water, dihydroxyacetone and glycerol. As several lactic acid bacteria have GlpFs in their lactate racemization operon (GlpF1/F4 phylogenetic group), their ability to transport this organic acid was tested. Both GlpF1 and GlpF4 facilitated the diffusion of D/L-lactic INTRODUCTION have more than two, with Pediococcus pentosaceus and Lc. lactis having four and Lactobacillus brevis three [9]. The existence of GLPs in these organisms may reflect their ability to use glycerol or other substrates in the presence of a second carbon source, since they cannot utilize glycerol alone [9]. However, the large number of MIPs in lactic acid bacteria suggests functions other than water and glycerol transport. Interestingly, a few MIPs have been shown to transport lactic acid in humans (AQP9), Arabidopsis [NIP2;1 (Nodulin26-like intrinsic protein)] and trematodes (SmAQP) [10– 12], suggesting that MIPs can facilitate the diffusion of lactic acid across membranes. Lactic acid, a monocarboxylic acid, and the lactate anion are in chemical equilibrium at a pK a of 3.86. Although the dissociated (ionized) lactate is the predominant species, the nondissociated (uncharged) lactic acid is always present at physiological pH. Unlike lactate, which requires protein transmembrane transport systems, lactic acid is able to diffuse through lipid bilayers [13]. However, the high lactate concentrations measured in lactic acid bacteria, such as Lactobacillus plantarum, show that some membranes are rather poorly permeable to lactic acid [14]. This implies that lactic acid transport may be regulated and that this regulation is a major factor in determining the cellular lactate concentration. Until now, no lactic acid channel has been functionally characterized in lactic acid bacteria. Lactic acid transport is often linked to energy production. The different energy-producing systems include lactate–proton symport [15], malolactic fermentation [16,17] and citrolactic fermentation [18]; in the last two cases, non-facilitated passive diffusion [19] of lactic acid is conceivable in addition to protein-mediated transport [20]. MIPs (major intrinsic proteins) facilitate the movement of water and non-ionic solutes across membranes and are required for osmoregulation, water conductance, gas and nutrient uptake and translocation, metalloid homoeostasis, and signal transduction in eubacteria, archaea, fungi, plants and animals. MIPs have been classified into two phylogenetically functional subgroups, AQPs (aquaporins), which originally consisted of water-specific channels, but now include channels shown to be permeable to other small uncharged solutes, and aquaglyceroporins, or GLPs (glycerol facilitators), which are permeable to glycerol and urea, with some also being permeable to water and metalloids [1]. Although the substrate specificity and roles of many plant and mammalian MIPs have been thoroughly studied [2], less is known about the physiological functions of MIPs in micro-organisms. Most of the known bacterial AQP-type isoforms have been identified in Gram-negative bacteria, whereas most of the GLP-type sequences have been found in Gram-positive ones. This asymmetry might be related to the different structures and diffusion properties of the membranes and cell walls in the two bacterial groups [3]. The best studied microbial MIPs are GlpF and AqpZ from Escherichia coli (EcGlpF and EcAqpZ) [4,5], GlpF from Lactococcus lactis (Gla) [6] and AqpX from Brucella abortus [7]. Phylogenetic analysis suggested that osmoregulation in eubacteria requires either one AQP and one GLP or a single GLP transporting both water and glycerol [3]. Some bacteria do not have any MIPencoding genes in their genome [8], but some lactic acid bacteria acid. Deletion of glpF1 and/or glpF4 in Lb. plantarum showed that both genes were involved in the racemization of lactic acid and, in addition, the double glpF1 glpF4 mutant showed a growth delay under conditions of mild lactic acid stress. This provides further evidence that GlpFs contribute to lactic acid metabolism in this species. This lactic acid transport capacity was shown to be conserved in the GlpF1/F4 group of Lactobacillales. In conclusion, we have functionally analysed the largest set of bacterial MIPs and demonstrated that the lactic acid membrane permeability of bacteria can be regulated by aquaglyceroporins. Key words: aquaporin, bacterial aquaglyceroporin, GlpF, lactic acid transport, Lactobacillus plantarum. Abbreviations used: AQP, aquaporin; At, Arabidopsis thaliana ; dak, dihydroxyacetone kinase; Ec, Escherichia coli ; GLP/GlpF, glycerol facilitator; Lar, lactate racemase; Lp, Lactobacillus plantarum; Ls, Lb. sakei ; MIP, major intrinsic protein; MRS, de Man, Rogosa and Sharpe; NIP, Nodulin26-like intrinsic protein; NPA, asparagine-proline-alanine; P f , osmotic water permeability coefficient; Pp, Pediococcus pentosaceus ; rAQP9, rat AQP9; YNB, yeast nitrogen base; ZmPIP2;5, Zea mays plasma membrane intrinsic protein 2;5. 1 These authors contributed equally to this work. 2 Present address: Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany. 3 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]). c The Authors Journal compilation c 2013 Biochemical Society 560 G. P. Bienert and others In the present study, we cloned six MIPs from Lb. plantarum, analysed their substrate specificity and characterized their function, with a particular focus on GlpF1 and GlpF4, which were shown to be lactic acid transporters. These are the first bacterial MIPs demonstrated to transport lactic acid. MATERIALS AND METHODS prepared as described previously [24]. PCR was performed using Phusion high-fidelity DNA polymerase (Finnzymes) or PfuTurbo Cx Hotstart DNA polymerase (Stratagene) in a 2720 Thermal Cycler (Applied Biosystems). The primers used in the present study were purchased from Eurogentec or Eurofins MWG Operon and are listed in Supplementary Table S2 (at http://www.biochemj. org/bj/454/bj4540559add.htm). Strains, plasmids and growth conditions The strains and plasmids used in the present study are listed in Supplementary Table S1 (at http://www.biochemj.org/bj/454/ bj4540559add.htm). Plasmids were constructed in E. coli TOP10. Lb. plantarum was grown in MRS (de Man, Rogosa and Sharpe) broth (Difco Laboratories) at 28 ◦ C without shaking. When required, erythromycin (250 μg/ml for E. coli, 10 μg/ml for Lb. plantarum) or chloramphenicol (10 μg/ml for both E. coli and Lb. plantarum) was added to the medium. Solid agar plates were prepared by adding 2 % (w/v) agar to the medium. The urea complementation assay and H2 O2 toxicity growth assay were carried out as described previously [21,22]. For the urea complementation assay, the dur3 mutant YNVW1 Saccharomyces cerevisiae strain was transformed with the vector described above containing the test GLP gene and transformants were selected on synthetic medium A [2 % agar, 2 % glucose, pH 5.5, and 0.17 % YNB (yeast nitrogen base) without amino acids and ammonium (Difco)], supplemented with 1 mM arginine, and spotted on to synthetic medium A supplemented with 1 mM arginine or different concentrations of urea as the sole nitrogen source. For the H2 O2 toxicity growth and survival assays, the above-described yeast strains were grown on synthetic medium B [2 % agar, 2 % glucose and 0.17 % YNB without amino acids (Difco)] containing different concentrations of H2 O2 . For the lactic acid complementation assay, the Δjen1 S. cerevisiae mutant strain (Euroscarf: BY4741; Mata; his3D1; leu2D0; met15D0; ura3D0; YKL217w::kanMX4) was transformed with pRS426-pTPIu containing the test GLP gene and transformants, selected on synthetic medium B supplemented according to the auxotrophic requirements with histidine, methionine and leucine, and spotted on to synthetic medium B supplemented according to the auxotrophic requirements with histidine, methionine and leucine and with glucose or different concentrations of D/L-lactate as the sole carbon source. After 5– 11 days of incubation at 28 ◦ C, differences in growth and survival in the different assays were recorded. Two to three independent experiments were performed and gave consistent results. Growth rate measurement Lb. plantarum cells were grown in 96-well sterile microplates with a transparent bottom (Greiner) and the attenuance (D600 ) was measured every 10 min with a Varioskan Flash multimode reader (Thermo Scientific). Growth competition experiment Lb. plantarum wild-type and the double glpF1 glpF4 mutant were grown overnight in MRS medium, then MRS medium with or without 200 mM L-lactate was inoculated with an equal volume of the two strains, resulting in a final D600 of 0.002. Inoculation was performed every 12 h in order to keep the cells in the exponential phase (D600 <1.0). Cultures were plated every 24 h on MRS agar and 96 PCRs were performed on isolated colonies in order to determine the percentages of wild-type and double mutant. DNA techniques and transformation General molecular biology techniques were performed as in Sambrook et al. [23]. Electrocompetent Lb. plantarum cells were c The Authors Journal compilation c 2013 Biochemical Society Construction of plasmids for transport assays Primers (Supplementary Table S2) matching the different glpF sequences were used to PCR amplify the glpF ORFs (open reading frames) from genomic DNA isolated from the different bacterial strains. The PCR products were directionally subcloned using a uracil excision-based improved high-throughput USER cloning technique [25] into the USER-compatible Xenopus expression vectors pNB1u and pNB1YFPu (N-terminal fusion of YFP with the protein of interest) [26] or the yeast expression vector pRS426pTPIu (Supplementary Table S1). Construction of deletion mutants Construction of strains LR0003 (glpF1) and LR0004 (glpF4) expressing deletion mutants of the Lb. plantarum MIP-encoding genes was performed as described previously [24]. In order to obtain the double-mutant strain LR0005 (glpF1 glpF4), strain LR0004 (ΔglpF4) was used and glpF1 was deleted following the same procedures described above. The strains and plasmids are listed in Supplementary Table S1. The primers used to construct the deletion vectors and to validate the deletions are listed in Supplementary Table S2. In vitro RNA synthesis and oocyte transport assays Ready-to-use capped complementary RNAs encoding N-terminal YFP-tagged or non-tagged GlpFs were synthesized in vitro as described previously [27]. Xenopus laevis oocytes were isolated, defolliculated and injected, and the Pf (osmotic water permeability coefficient) was determined as described previously [27]. Localization of the YFP-tagged fusion proteins was verified by confocal microscopy (LSM 710; Carl Zeiss) on fixed oocytes as described previously [28]. YFP was excited at 514 nm and the emitted fluorescence detected between 530 and 570 nm. Glycerol, dihydroxyacetone or lactic acid permeability was measured in an iso-osmotic swelling assay as described previously [29] with the following adaptations. Prior to the experiment, the oocytes were osmotically equilibrated in standard Barth’s solution (200 mOsm/l) for 15–25 min. Solute uptake was tested by measuring the swelling rate at room temperature (20 ◦ C) by video microscopy on incubation in an iso-osmotic modified Barth’s solution (200 mOsm/l) in which NaCl was replaced with 160 mM glycerol, 80 mM 1,3-dihydroxyacetone dimer (corresponding to approximately 160 mM dihydroxyacetone in solution) or 80 mM sodium D-, L- or D/L-lactate (corresponding to approximately 58 μM non-dissociated lactic acid at pH 7.0). The swelling rate of the oocytes was expressed as the rate of change in oocyte volume [d(V/V 0 )/dt, where V is the volume at a specific time and V 0 the initial oocyte volume at zero time], as described previously [29]. Direct lactic acid uptake experiments in oocytes were performed as follows. Oocytes were incubated at room temperature for 0, 6 or 12 min in a modified Barth’s solution containing 1 mM unlabelled sodium lactate and 14 C-labelled sodium lactate (12 μCi/ml) (PerkinElmer) with the osmolarity adjusted to 200 mOsm/l with NaCl, then were washed four times with 250 ml of ice-cold modified Barth’s solution containing Aquaglyceroporins from Lb. plantarum 80 mM non-labelled sodium lactate, lysed overnight with 1 ml of 10 % (w/v) SDS, and the radioactivity was counted in a scintillation counter after addition of 4 ml of Lumasafe scintillation liquid (PerkinElmer). 561 Sequencing results showed that the database sequences were in agreement with the cloned sequences. All six Lb. plantarum GlpF (LpGlpF) sequences contained the typical selectivity filter residues and NPA (asparagine-proline-alanine) boxes of bacterial MIPs (Supplementary Table S5 and Supplementary Figure S1 at http://www.biochemj.org/bj/454/bj4540559add.htm). Lactate racemization assays Lb. plantarum cells were grown in MRS medium and Lar (lactate racemase) activity was induced by addition of L-lactate sodium salt (Sigma–Aldrich) at a final concentration of 200 mM during the mid-exponential phase (D600 = 0.6–0.7), then the cells were collected 4 h later, washed twice with 1 volume of 60 mM Mes buffer, pH 6 (R buffer), and resuspended in 1/20 volume of R buffer containing 0.17–0.18 mm glass beads (Sartorius Mechatronics Biomedicals). The cells were homogenized for 2× 1 min at 6.5 m/s in a FastPrep-24 (MP), the homogenate was centrifuged at 13 000 g for 15 min (4 ◦ C), and the supernatant was collected. The cytoplasmic Lar-specific activity was assayed by incubating the supernatant with 20 mM D- or L-lactate in R buffer at 35 ◦ C for 10 min, stopping the reaction by incubation for 10 min at 90 ◦ C, and measuring the conversion to L- or D-lactate using the D-lactic acid/L-lactic acid UV test (R-Biopharm). The protocol was adapted to 100 μl reaction volumes in 96-well, halfarea microplates (Greiner). Total protein in the supernatant was measured according to the method of Bradford [30], using the BioRad Laboratories protein assay and the cytoplasmic Lar-specific activity expressed as μmol of D- or L-lactate produced per min per mg of total protein. Whole-cell Lar activity was measured in cell suspensions (D600 of 1.0) of Lar-induced Lb. plantarum, prepared as described previously [31], but using 50 mM D- or L-lactate instead of glucose; the cell suspension was incubated for 1 h at 30 ◦ C, then L- or D-lactate formation was measured as above and whole-cell Lar activity was expressed as μmol of lactate produced per h by 1 ml of cells. Gene context analysis and phylogenetic alignment Genes surrounding glpF homologues of Lb. plantarum WCFS1 were retrieved from the NCBI database (release 189) and are listed in Supplementary Table S3 (at http://www.biochemj.org/ bj/454/bj4540559add.htm). The MIP protein sequences for all other Lactobacillales were retrieved from the NCBI database (release 189) and are listed in Supplementary Table S4 (at http:// www.biochemj.org/bj/454/bj4540559add.htm). They were aligned using ClustalX2 [32] and the phylogenetic tree was constructed using the neighbour-joining method [33]. The tree was visualized using treeGraph2 [34]. RESULTS In silico analysis and cloning of Lb. plantarum MIPs The available genomes for Lb. plantarum WCFS1, Lb. plantarum JDM1 and Lb. plantarum subsp. plantarum ST-III were screened for MIP-encoding genes and six were identified in each of the three genomes by gene annotation and homology search and annotated as glpF1, glpF2, glpF3, glpF4, glpF5 and glpF6. The deduced lengths of the different GlpF proteins ranged from 216 to 250 amino acids and the deduced protein sequences for a given GlpF in the three different bacterial strains were 100 % identical (Supplementary Figure S1 at http://www.biochemj.org/bj/454/ bj4540559add.htm). Using gene-specific primers based on the genomic data, MIP-encoding ORFs were amplified by PCR from the genomic DNA of Lb. plantarum WCFS1 and cloned. Phylogenetic and genetic context analysis of Lactobacillales MIPs To predict the role of glpF genes from Lb. plantarum, we performed a BLAST comparison of EcGlpF against Lactobacillales genomes. The homologues from 17 species representative of the diversity within this order of Gram-positive bacteria were retained. A total of 49 MIP-encoding genes were found in these species and their number in each species ranged from one to six (denoted 1–6 in Supplementary Table S4). The encoded proteins, together with EcAqpZ, EcGlpF, and human Aqp0 as an outgroup, were aligned with ClustalX2 [32] and a phylogenetic tree constructed using the neighbour-joining method [33]. Five distinct phylogenetic groups were deduced from this alignment (Figure 1A). In order to infer a potential role of each of these groups and of the six glpF genes from Lb. plantarum, we looked at their genetic context (Figure 1B). The first group (named AqpZ) was composed of E. coli AqpZ and LpGlpF6 and their homologues. No function could be attributed to this group on the basis of their genetic context (Supplementary Table S3), but, owing to their homology with EcAqpZ, we hypothesize that it is composed of water channels. LpGlpF1 and LpGlpF4 clustered together in the second group (named GlpF1/F4) as expected, since their protein sequences were 92 % identical. The glpF1 gene of Lb. plantarum is part of the lar operon, consisting of larA, larB, larC, larD (glpF1) and larE (Figure 1B). Since glpF1 is located in this operon, in which all the other genes have been shown to be involved in the lactate racemization (Lar) activity of the bacterium [35], this suggests it plays a direct role in lactic acid transport. Four of the ten homologues in the GlpF1/F4 group were also found in lactate racemization operons (lar operons), suggesting that this group consists of lactic acid channels or proteins involved in a transport process contributing to the Lar activity. The third group (named GlpF2) was composed of LpGlpF2 homologues. The glpF2 gene of Lb. plantarum and several LpGlpF2 homologues are part of an operon composed of dak1B (dihydroxyacetone kinase 1B), dak2 and dak3 that is similar to the operon coding for the dak system in Lc. lactis [36] (Figure 1B). Since dihydroxyacetone is chemically very similar to glycerol, and since other mammalian and protozoan GLPs have been demonstrated to transport this molecule [37], it is likely that LpGlpF2 is a dihydroxyacetone transporter. The fourth group (named GlpF3) was composed of LpGlpF3 homologues. Several of these (including Lb. plantarum glpF3) have been assigned to operons composed of glpK and glpD genes, which are similar to those responsible for glycerol utilization in E. coli [38] (Figure 1B), so we hypothesized that glpF3 encoded a glycerol transporter. Two exceptions were found, these being Lactobacillus salivarius GlpF3 and S. agalactiae GlpF3, which clustered, respectively, with dihydroxyacetone (GlpF2) or glycerol (GlpF3) transporters, but whose genetic context pointed respectively to glycerol or dihydroxyacetone transport (Figure 1A). It seems that the dihydroxyacetone and glycerol channels are functionally very closely related and that interconversion of one into the other may occur during evolution. The fifth group (named GlpF5) was composed of LpGlpF5 homologues and homologues of Gla, the aquaglyceroporin of Lc. lactis, which has been shown to transport both water and glycerol c The Authors Journal compilation c 2013 Biochemical Society 562 Figure 1 G. P. Bienert and others Phylogenetic analysis and phylogenetic context of Lb. plantarum GlpFs (A) Phylogenetic tree of 49 MIPs from 17 Lactobacillales species, GlpF and AqpZ from E. coli , and Aqp0 from Homo sapiens . The number after the species name corresponds to that used for this isoform in this species in Supplementary Table S4 (at http://www.biochemj.org/bj/454/bj4540559add.htm). The relevant genetic context of the MIP-encoding genes is given in parentheses as Lar (lactate racemization operon), Dha (dihydroxyacetone utilization operon), Gly (glycerol utilization operon) or Prop (propanediol utilization operon). The GlpFs that were functionally investigated in the present study are shown in bold. En. faecalis, Enterococcus faecalis ; G. adiacens , Granulicatella adiacens ; Le ., Leuconostoc ; S., Streptococcus ; W. koreensis , Weissella koreensis . (B) Genetic context of each of the six glpF genes from Lb. plantarum WCFS1. The glpF1–glpF6 genes are shown in white background and the other genes in black background. For details of gene annotation, see Supplementary Table S3 (at http://www.biochemj.org/bj/454/bj4540559add.htm). [6]. However, two members of this GlpF5 group were also found to be located in the lar operon, indicating a potential role in lactic acid transport (Figure 1A and Supplementary Table S4). c The Authors Journal compilation c 2013 Biochemical Society The glpF4, glpF5 and glpF6 genes of Lb. plantarum are not part of any operon and their putative function cannot be deduced from their genetic context (Figure 1B and Supplementary Table S3). Aquaglyceroporins from Lb. plantarum Figure 2 563 Water, glycerol and dihydroxyacetone membrane permeability of X. laevis oocytes expressing Lb. plantarum GlpFs (A) P f values of oocytes injected with water (negative control) or cRNA encoding rAQP9 (positive control) or the indicated LpGlpF isoform. The swelling rates were determined on immersion for 70 s in half-strength Barth’s solution. The data are expressed as the means of measurements for 11–14 oocytes in one experiment and are representative of those obtained in three separate experiments. (B) Glycerol permeability of oocytes injected with water or cRNA encoding rAQP9 (positive control), ZmPIP2;5 (negative control), LpGlpF2, LpGlpF3 or LpGlpF4, or co-injected with cRNAs encoding ZmPIP2;5 and LpGlpF1, LpGlpF5 or LpGlpF6. The initial swelling rates were determined on immersion for 170 s in iso-osmotic Barth’s solution containing 160 mM glycerol. The data are expressed as the means of measurements for 8–11 oocytes in one experiment and are representative of those obtained in three separate experiments. (C) Dihydroxyacetone permeability of oocytes injected with water or cRNA encoding rAQP9 (positive control), ZmPIP2;5 (negative control), LpGlpF2, LpGlpF3 or LpGlpF4 or co-injected with cRNAs encoding ZmPIP2;5 and LpGlpF1, LpGlpF5 or LpGlpF6. The initial swelling rates were determined as described in (B) by immersion for 170 s in 160 mM dihydroxyacetone (35 s for rAQP9). The data are expressed as the means of measurements on 20–33 oocytes in two independent experiments. In all experiments, 25 ng of cRNA for each MIP-encoding gene was used, except in the case of ZmPIP2;5 (2 ng). V and V 0 indicate the volume at a given time point and the initial volume respectively. The error bars are the 95 % confidence intervals. Water, glycerol and dihydroxyacetone permeability of Lb. plantarum GlpFs The water channel activity of Lb. plantarum GlpFs was determined by heterologous expression in X. laevis oocytes and swelling assays in a hypo-osmotic medium. Water-injected oocytes (negative control) or oocytes expressing the respective GlpF isoform (LpGlpF1–LpGlpF6) were suspended in halfstrength Barth’s solution, which resulted in an outwardly directed osmotic gradient of 100 mOsm. As shown in Figure 2(A), the water-injected oocytes had a low Pf owing to limited transmembrane water movement, whereas cells expressing the positive control rAQP9 (rat AQP9), a well-characterized mammalian GLP protein, had a high Pf [39]. No significant increase in Pf was detected for cells injected with LpGlpF1, LpGlpF5 or LpGlpF6 cRNA, whereas the Pf value of oocytes injected with LpGlpF2, LpGlpF3 or LpGlpF4 cRNA was approximately 4-, 6- or 7-fold higher respectively than that of the water-injected oocytes. To determine whether the lack of a Pf increase in oocytes expressing LpGlpF1, LpGlpF5 or LpGlpF6 was due to a problem of protein expression or trafficking, oocytes were injected with cRNAs encoding the GlpFs fused to YFP (YFP–LpGlpF1 to YFP–LpGlpF6) and the localization of the YFP-tagged proteins was analysed using confocal microscopy. As shown in Supplementary Figure S2 (at http:// www.biochemj.org/bj/454/bj4540559add.htm), no specific fluorescent signal was detected in water-injected oocytes, whereas all six sets of YFP–LpGlpF-expressing cells showed a similar fluorescence distribution and intensity at their periphery, indicating a plasma membrane localization, but, in contrast with the positive control YFP–ZmPIP2;5 (Zea mays plasma membrane intrinsic protein 2;5), an additional strong internal YFP signal was detected with the Lb. plantarum GlpFs. These results suggest that all Lb. plantarum isoforms are expressed in oocytes and are, at least partially, targeted to the plasma membrane. The glycerol permeability of the LpGlpF isoforms was measured in an iso-osmotic swelling assay with a 160 mM glycerol inwardly-directed chemical gradient. Under these conditions, glycerol enters the cells due to the chemical concentration difference, resulting in a secondary osmotic gradient and an influx of water. To observe cell swelling, the membrane permeability for water must be sufficiently high; this is the case if the channel of interest is also permeable to water or if the water-impermeable MIP is co-expressed with a channel that is water-permeable, but impermeable to the solute tested [29,40]. Oocytes were injected with cRNA encoding the waterpermeable LpGlpF2, LpGlpF3 or LpGlpF4 isoform or co-injected with cRNAs encoding LpGlpF1, LpGlpF5 or LpGlpF6 and ZmPIP2;5, a water-permeable and glycerol-impermeable plant AQP [41]. As shown in Figure 2(B), oocytes injected with water or expressing ZmPIP2;5 alone did not swell during the monitored time period, whereas oocytes expressing LpGlpF2 or LpGlpF4 showed significantly high swelling rates, similar to those of cells expressing the positive control rAQP9, and LpGlpF3-expressing oocytes displayed an even higher swelling rate. Co-expression of LpGlpF1, LpGlpF5 or LpGlpF6 with ZmPIP2;5 did not result in any volume increase. To verify that co-expression of ZmPIP2;5 with LpGlpFs did not modify its water permeability, the Pf of oocytes expressing ZmPIP2;5 alone or together with LpGlpF1, LpGlpF5 or LpGlpF6 was measured and oocytes co-expressing the two channels were found to have a significantly higher Pf than water-injected oocytes, similar to that of oocytes expressing only ZmPIP2;5 (Supplementary Figure S3A at http://www. biochemj.org/bj/454/bj4540559add.htm), demonstrating that the water channel activity of ZmPIP2;5 was not impaired when coexpressed with GlpF proteins and that the lack of detected glycerol permeability was not due to a restrictive water permeability of the oocyte membrane. The facilitated diffusion of dihydroxyacetone through LpGlpF isoforms was analysed in an iso-osmotic swelling assay in the presence of a 160 mM inwardly-directed chemical gradient. c The Authors Journal compilation c 2013 Biochemical Society 564 G. P. Bienert and others As shown in Figure 2(C) and as previously shown [37], rAQP9 was a highly efficient dihydroxyacetone channel. In addition, the swelling rate of oocytes expressing LpGlpF2, LpGlpF3 or LpGlpF4 was significantly increased after transfer to dihydroxyacetone-containing buffer. Compared with the waterinjected cells, the volume of oocytes expressing ZmPIP2;5 alone or co-expressing ZmPIP2;5 with LpGlpF1, LpGlpF5 or LpGlpF6 increased only slightly. As this increase was small and was also observed for ZmPIP2;5, it could result from influx of water via ZmPIP2;5 following the non-facilitated transmembrane diffusion of dihydroxyacetone. The water and dihydroxyacetone permeability studies shown in Supplementary Figure S3(B) and Figure 2(C) were performed on the same batches of oocytes and the results therefore strongly suggest that the increased swelling rate seen in oocytes expressing LpGlpF2, LpGlpF3 or LpGlpF4 was due to dihydroxyacetone permeability and not to their intrinsic water permeability. Indeed, oocytes expressing these three channels had much lower Pf values than ZmPIP2;5expressing oocytes (Supplementary Figure S3B), whereas a much greater swelling rate was seen in dihydroxyacetone-containing buffer (Figure 2C). Taken together, these swelling assay results show that LpGlpF2, LpGlpF3 and LpGlpF4 are each permeable to water, glycerol and dihydroxyacetone, whereas no transport was seen with LpGlpF1, LpGlpF5 or LpGlpF6. Urea and H2 O2 permeability of Lb. plantarum GlpFs Lb. plantarum GlpFs were tested for urea permeability in a complementation assay by heterologous expression in the yeast mutant strain YNVW1 (dur3). This strain is deficient for the yeast intrinsic urea transporter DUR3 and grows only on medium supplemented with high concentrations of urea as the sole nitrogen source or with an alternative nitrogen source (i.e. arginine) [42]. As shown in Figure 3(A), expression of LpGlpF1 or LpGlpF4 rescued yeast growth on medium containing low concentrations of urea (1, 2 or 3 mM), as also seen with the positive control, the urea/H + symporter AtDUR3 (where At is Arabidopsis thaliana). In contrast, none of the other GlpFs from Lb. plantarum were able to facilitate uptake of urea into yeast cells. The results of this complementation assay show that LpGlpF1 and LpGlpF4 facilitate urea diffusion. The H2 O2 permeability of LpGlpFs was investigated in the same yeast mutant used for the urea complementation assay. Yeast cells expressing LpGlpFs were exposed to different H2 O2 concentrations in the growth medium and survival was monitored on plates. As shown in Figure 3(B), expression of LpGlpF1, LpGlpF3 or LpGlpF4 resulted in complete growth inhibition at 0.8 mM H2 O2 , whereas yeast cells carrying an empty vector (control) or expressing the urea/H + symporter AtDUR3, LpGlpF5 or LpGlpF6 grew normally. LpGlpF2 showed an intermediate sensitivity to H2 O2 and displayed complete growth inhibition at 1.2 mM H2 O2 . The results of this toxicity assay show that LpGlpF1, LpGlpF3, LpGlpF4 and, possibly, LpGlp2 are permeable to H2 O2 . Lactic acid permeability of Lb. plantarum GlpFs As (i) Lb. plantarum belongs to the lactic acid-producing bacteria, (ii) LpGlpF1 is located in a lactic acid-responsive operon and (iii) a few other MIPs facilitate lactic acid diffusion, the lactic acid channel activity of LpGlpFs was investigated in a complementation assay by heterologous expression in the yeast mutant strain jen1. Disruption of Jen1p abolishes uptake of lactate into yeast cells [43] and prevents their growth on medium c The Authors Journal compilation c 2013 Biochemical Society Figure 3 Urea and H2 O2 transport assays in yeast expressing Lb. plantarum GlpF isoforms (A) Urea transport complementation assay for LpGlpF isoforms. The YNVW1 (dur3 ) yeast mutant defective in urea uptake [21] transformed with the empty vector pRS426-pTPIu (negative control) or pRS426-pTPIu containing the indicated LpGlpF gene or AtDUR3 (positive control) at a D 600 of 1, 0.01 or 0.0001 was spotted on to medium containing various concentrations of urea or arginine as the sole nitrogen source and growth was recorded after 10 days at 28 ◦ C. The results shown are for yeast spotted at a D 600 of 1; a similar sensitivity was seen in the other two sets of cultures. Control indicates the empty vector. (B) H2 O2 sensitivity of yeast cells expressing Lb. plantarum glpF s. Cultures of dur3 yeast cells transformed with the empty vector pRS426-pTPIu (control) or pRS426-pTPIu carrying the indicated LpGlpF isoform or AtDUR3 at a D 600 of 1, 0.01 and 0.0001 were spotted on to medium containing the indicated concentrations of H2 O2 and growth was recorded after 6 days at 28 ◦ C. The growth behaviour and survival rates of the different transformants is shown for the yeasts spotted at a D 600 of 0.01; a similar sensitivity was seen in the other two sets of cultures. All yeast growth assays were performed at least twice, with consistent results. containing low amounts of lactic acid/lactate as the sole carbon source and only transformation with a plasmid encoding a protein able to transport lactic acid/lactate allows this mutant strain to grow in this medium. As shown in Figure 4(A), expression of LpGlpF1 or LpGlpF4 rescued yeast growth on medium containing low concentrations of D/L-lactate (0.1, 0.25 or 0.5 % w/v), but none of the other LpGlpFs were able to complement the growth of jen1 strain under these conditions. However, all GlpF-expressing strains grew on an alternative carbon source, glucose. It was interesting to observe that GlpF1- and GlpF4expressing strains grew less than the other strains on glucose medium, but rescued the growth on medium containing D/Llactate. This complementation assay showed that LpGlpF1 and LpGlpF4 facilitate lactic acid diffusion. To further investigate the lactic acid channel activity of LpGlpFs, an oocyte iso-osmotic swelling assay using a 80 mM sodium D/L-lactate inwardly-directed chemical gradient was performed. rAQP9 was used as a positive control, as it facilitates lactic acid diffusion [44]. Neither the co-expression of LpGlpF1, LpGlpF5 or LpGlpF6 with ZmPIP2;5, nor the expression of LpGlpF2 and LpGlpF3 alone, led to an significant increase in the oocyte swelling rate compared with the water- and ZmPIP2;5 cRNAinjected cells (Figure 4B and Supplementary Figure S4 at http://www.biochemj.org/bj/454/bj4540559add.htm). In contrast, oocytes expressing LpGlpF4 or the positive control rAQP9 showed a 330- and 750-fold higher swelling rate respectively, showing that LpGlpF4 is a functional lactic acid channel (Figure 4B and Supplementary Figure S4). To confirm these results in an independent experiment, we directly measured [14 C]lactic acid/lactate accumulation in oocytes expressing LpGlpF1, LpGlpF4 or rAQP9 as a positive control after different incubation times in 12 μCi/ml sodium [14 C]lactate. As shown in Figure 4(C), oocytes expressing LpGlpF4 or rAQP9 accumulated Aquaglyceroporins from Lb. plantarum Figure 4 565 Lactic acid membrane permeability of X. laevis oocytes and S. cerevisiae cells expressing Lb. plantarum GlpF isoforms (A) Lactic acid transport complementation assay of LpGlpF isoforms. Cultures of Δjen1 yeast mutants defective in lactate uptake capacity [43] transformed with the empty vector pRS426-pTPIu (negative control) or pRS426-pTPIu containing the indicated LpGlpF gene at a D 600 of 1, 0.1 and 0.01 were spotted on to medium containing the indicated concentration of D/L-lactate or 2 % glucose as the sole carbon source and growth was recorded after 10 days at 28 ◦ C. (B) D/L-lactic acid membrane permeability of oocytes injected with water (negative control) or cRNA encoding rAQP9 (positive control), LpGlpF2, LpGlpF3 or LpGlpF4 or co-injected with cRNAs coding for ZmPIP2;5 and LpGlpF1, LpGlpF5 or LpGlpF6. The initial swelling rates were determined on immersion for 350 s in iso-osmotic Barth’s solution containing 80 mM D/L-lactate. The results are the means of measurements of 8–12 oocytes in a single experiment and are representative of those obtained in three separate experiments. The error bars are the 95 % confidence intervals. (C) Lactic acid uptake by oocytes injected with water or cRNA encoding rAQP9, LpGlpF1 or LpGlpF4. Oocytes were incubated for the indicated time in modified Barth’s solution supplemented with 12 μCi/ml sodium [14 C]lactate and the lactate/lactic acid content was determined as described in the Materials and methods section. The results are expressed as the means of measurements of 16–20 oocytes in one experiment and are representative of those obtained in three separate experiments. The error bars are the 95 % confidence intervals. (D) D- or L-lactic acid membrane permeability of oocytes injected with water (negative control) or cRNA coding for LpGlpF4. The initial swelling rates were determined as in (B) by immersion for 350 s in 80 mM D/L-lactic acid (open bars), 80 mM D-lactic acid (light grey bars) or 80 mM L-lactic acid (dark grey bars). The results are expressed as the means of measurements for four (D-/L-lactic acid) or 10–12 (D- or L-lactic acid) oocytes in a single experiment and are representative of those obtained in three experiments. The error bars represent the 95 % confidence intervals. V and V 0 indicate the volume at a given time point and the initial volume respectively. approximately twice as much lactic acid/lactate as water-injected control oocytes when incubated for 6 or 12 min with 12 μCi/ml sodium [14 C]lactate, confirming that LpGlpF4 is an efficient lactic acid transporter in the oocyte system. In contrast, expression of LpGlpF1 resulted in similar lactic acid/lactate uptake to that seen with water-injected control oocytes. Stereoselectivity of LpGlpF4 for lactic acid E. coli GlpF shows stereospecificity for the transport of substrates with different chain lengths and chiralities, which influence the hydrophobic contacts between the transported solute and the channel residues [45]. Lactic acid has two optical isomers, L and D, which are both synthesized and interconverted in Lb. plantarum [35]. To test whether LpGlpF4 showed stereoselectivity for one of the two optical isomers, its channel activity for L-lactic acid, Dlactic acid and a D/L racemic mixture were compared in the oocyte iso-osmotic swelling assay. As shown in Figure 4(D), LpGlpF4expressing oocytes showed a similar increase in swelling rate under the three different conditions, showing that the D- and Llactic acid isomers were equally well transported by this protein. Transport selectivity of GlpF1/F4 homologues from P. pentosaceus and Lactobacillus sakei To investigate the transport selectivity of GLPs from other lactic acid bacteria belonging to the same phylogenetic GlpF1/F4 group as LpGlpF1 and LpGlpF4 and located in a lactate racemization operon (Supplementary Table S4), the glpF1 orthologues of P. pentosaceus and Lb. sakei were cloned and the corresponding proteins were tested for water, glycerol and lactic acid permeability by heterologous expression in oocytes as described above. As shown in Figure 5, expression of PpGlpF1 (where Pp is P. pentosaceus) and LsGlpF1 (where Ls is Lb. sakei) induced a significant increase in the oocyte membrane permeability for all three tested compounds, demonstrating that GlpF1 orthologues facilitate the diffusion of commonly tested solutes and that PpGlpF1 and LsGlpF1 are functional lactic acid channels. LpGlpF1 and LpGlpF4 transport lactic acid in Lb. plantarum WCFS1 To confirm the lactic acid channel activity of LpGlpF4 in bacterial cells, three marker-free deletion mutant strains (glpF1, glpF4 c The Authors Journal compilation c 2013 Biochemical Society 566 Figure 5 G. P. Bienert and others Transport characteristics of X. laevis oocytes expressing glpF1 orthologues from P. pentosaceus and Lb. sakei (A) P f values. (B) Glycerol permeability. (C) D/L-lactic acid permeability. Oocytes were injected with water (negative control) or cRNA encoding rAQP9 (positive control), PpGlpF1 or LsGlpF1. The amount of injected cRNA and the measurement of the initial swelling rate were identical to those in Figures 2 and 4. Mean values are shown for (A) a total of 14 water-injected oocytes, 16 AQP9-injected oocytes, 18 PpGlpF-injected oocytes and 17 LsGlpF-injected oocytes in two independent experiments; (B) a total of 14 water-injected oocytes, 13 AQP9-injected oocytes, 11 PpGlpF-injected oocytes and 12 LsGlpF-injected oocytes in two independent experiments; (C) a total of 14 water-injected oocytes, 13 AQP9 injected-oocytes, 16 PpGlpF-injected oocytes, and 14 LsGlpF-injected oocytes in two independent experiments. The error bars represent the 95 % confidence intervals. and glpF1 glpF4) were generated in Lb. plantarum WCFS1 and assayed for lactic acid transport by incubating non-growing cells with 50 mM D- or L-lactate and monitoring L- or Dlactate production. This whole-cell lactate racemization (Lar) activity was standardized to the cytoplasmic Lar-specific activity measured in the supernatant of cell homogenates, giving the whole-cell Lar activity/cytoplasmic Lar specific activity ratio, which is used as a means of evaluating the transport of lactic acid across cell membranes. As shown in Figure 6(A), at pH 6.5, both of the single mutant strains showed decreased lactic acid transport ability expressed as a percentage of that for the D- to L-lactate conversion in the wild-type, whereas the double mutant strain showed an even greater decrease, which corresponded to the additive effect of the two simple inactivations. When the pH of the medium was reduced to pH 5.5 or increased to pH 7.5, the lactic acid transport ability of the wild-type and all three types of mutant cells was enhanced or reduced to the same extent, showing that the transported molecule was lactic acid and not the lactate anion (Figure 6B shows the result for the wild-type). This is in agreement with the assumption that, with very few exceptions, MIPs only facilitate the transport of uncharged solutes. These results clearly show that both GlpF1 and GlpF4 transport lactic acid in Lb. plantarum WCFS1. Physiological importance of lactic acid transport As shown in Figure 7(A), when wild-type Lb. plantarum and the double mutant strain (glpF1 glpF4) were grown in MRS medium, the double mutant showed a growth delay during the exponential growth phase that was even more marked when the medium was supplemented with 200 mM L-lactate, suggesting that the double mutant had an increased sensitivity to mild lactic acid stress. To determine whether the growth delay affected the fitness of the double mutant compared with the wild-type, coculture (1:1 ratio) competition experiments were performed in which the two strains were grown in MRS medium with or without 200 mM L-lactate and kept in exponential phase for three days, then the wild-type/mutant ratio was assessed by PCR on isolated colonies. As shown in Figure 7(B), the wild-type clearly prevailed over the mutant after three days of exponential growth (> 90 % of the global population) and, under mild lactic acid c The Authors Journal compilation c 2013 Biochemical Society stress conditions, this effect was even more striking, since none of the 96 colonies tested after three days growth in MRS medium plus 200 mM L-lactate contained the mutant strain. These data show that the double mutant had a reduced fitness, probably due to its reduced lactate transport capability, and suggests that, under lactic acid stress, it was even less efficient in excreting the lactic acid it produced, resulting in an even lower fitness under these conditions. Interestingly, the mutant was able to grow to a higher attenuance than the wild-type in medium with or without added lactate (Figure 7A). Lactic acid is known to have an inhibitory effect on growing cells, especially at low pH [46], and this inhibitory effect would be mitigated in cells showing limited lactic acid transport, giving the mutant a growth advantage at the end of the exponential phase, when medium acidification becomes inhibitory. DISCUSSION MIPs are a large family of channel proteins that are present in all kingdoms of life. Although higher eukaryotes have a high number of MIP isoforms, micro-organisms have either only a few (e.g. two and four in E. coli and S. cerevisiae respectively) or none at all, as is the case in many bacteria [8]. This can be explained by the absence of membrane-surrounded subcellular organelles and/or the high surface-to-volume ratio, which makes, in theory, channel-mediated membrane diffusion optional. The present study identified a number of MIP-encoding genes in the order Lactobacillales, which includes all lactic acid bacteria species. Interestingly, six of these were found to be present in Lb. plantarum, a highly adaptable and versatile species, which is found in a variety of environmental niches in which the different growth conditions require different transporters. This number of MIP-encoding genes is the highest so far reported for any bacterial strain. The phylogenetic analysis of 49 MIPs from diverse Lactobacillales species, including Lb. plantarum, identified five different MIP groups, each containing at least one Lb. plantarum isoform, underlining the potential flexibility of this species in its use of different solutes. The analysis of the genetic context revealed that three MIP-encoding genes were located in operons involved in the metabolism of glycerol (LpGlpF3), Aquaglyceroporins from Lb. plantarum Figure 6 567 Lactic acid transport assay of Lb. plantarum cells deficient in GlpF1 and/or GlpF4 (A) Bidirectional lactic acid racemization (Lar) assay of cell suspensions of Lb. plantarum WCFS1 (WT), mutant glpF1 (1), mutant glpF4 (4) or the double mutant glpF1 glpF4 (14) at pH 6.5. The whole-cell Lar activity/cytoplasmic Lar-specific activity ratio was calculated and expressed as a percentage of that for the D- to L-lactate conversion by Lb. plantarum WCFS1 cells. Mean values for four experiments are shown. (B) Bidirectional Lar assay of cell suspensions of Lb. plantarum WCFS1 (WT) at pH 7.5, pH 6.5 and pH 5.5. The whole-cell Lar activity/cytoplasmic Lar-specific activity ratio was calculated for each pH value and expressed as a percentage of that for the D- to L-lactate conversion at pH 6.5. Mean values for three experiments are shown. The error bars represent the 95 % confidence intervals. Figure 7 Growth and fitness of Lb. plantarum cells deficient in GlpF1 and GlpF4 (A) Growth curves of wild-type (WT) Lb. plantarum WCFS1 (continuous lines) and its isogenic mutant ΔglpF1 ΔglpF4 (broken lines) in MRS medium (black lines) or MRS supplemented with 200 mM L-lactate (grey lines). The mean value for 16 individual cultures is shown. (B) Competition experiment between the WT and double mutant (initial ratio 1:1) in MRS medium (black bars) or MRS supplemented with 200 mM L-lactate (grey bars). The percentage of the double mutant in the whole population was assessed by PCR on 96 individual colonies from three independent cultures (32 each) after inoculation (day zero) and after 1, 2 or 3 days. The error bars represent the 95 % confidence intervals. dihydroxyacetone (LpGlpF2) and lactic acid (LpGlpF1). Transport assays performed in Xenopus oocytes, S. cerevisiae cells and Lb. plantarum deletion mutants confirmed the substrate predictions based on the genetic context analysis. Although Lb. plantarum GlpFs were found to be mixed-solute channels, LpGlpF2, LpGlpF3 and LpGlpF1 transported dihydroxyacetone, glycerol and lactic acid respectively in addition to other solutes (see Table 1 for a summary). The present study highlights the fact that the determination of the genetic context of MIP-encoding genes in bacterial genomes helps identify their substrates and elucidates their physiological function. No substrate could be identified for LpGlpF5 and LpGlpF6, even though the homology of LpGlpF6 with EcAqpZ suggests a water channel activity. The GlpF5 group appeared more diverse and no clear transport specificity could be assigned to this group, since some members transport glycerol and water [6] and others are found in lar operons, suggesting an involvement in lactic acid transport. The absence of transport activity when LpGlpF1, LpGlpF5 or LpGlpF6 was expressed in oocytes is intriguing, since they seem to be, at least partly, targeted to the plasma membrane, as shown by the use of YFP-tagged variants (Supplementary Figure S2). One possible explanation might be that they are non-functional in this heterologous host membrane, as reported for other microbial AQPs [47]. Whether this is due to a problem of protein conformation, selectivity or regulation remains to be determined. Even though LpGlpF1 appeared to be non-functional in oocytes, it was shown to facilitate the passage of lactic acid, urea and H2 O2 across membranes when heterologously expressed in yeast and to contribute to lactic acid metabolism in Lb. plantarum (Table 1). To shed light on the mechanisms and structural requirements determining substrate selectivity in bacterial MIPs, we tested their permeability to urea and H2 O2 in the yeast system. Like water and glycerol, H2 O2 and urea are typically transported by aquaporins or aquaglyceroporins respectively. The best-characterized bacterial c The Authors Journal compilation c 2013 Biochemical Society 568 Table 1 G. P. Bienert and others Summary of the substrate permeability of GlpFs from Lb. plantarum , Lb. sakei and P. pentosaceus + , transport; + / − , transport; ND, no transport detected; NT, the substrate was not tested. The type of assay used is indicated in parentheses as O (uptake assay in oocytes), B (bacterial cell racemization assay) or Y (yeast complementation or toxicity growth assay). Tested substrate and functional assay system GlpF isoform Water Glycerol Dihydroxyacetone Lactic acid Urea H2 O2 LpGlpF1 LsGlpF1 PpGlpF1 LpGlpF2 LpGlpF3 LpGlpF4 LpGlpF5 LpGlpF6 ND (O) + (O) + (O) + (O) + (O) + (O) ND (O) ND (O) ND (O) + (O) + (O) + (O) + (O) + (O) ND (O) ND (O) ND (O) NT NT + (O) + (O) + (O) ND (O) ND (O) + (Y, B) + (O) + (O) ND (Y, O) ND (Y, O) + (Y, O, B) ND (Y, O) ND (Y, O) + (Y) NT NT ND (Y) ND (Y) + (Y) ND (Y) ND (Y) + (Y) NT NT + / − (Y) + (Y) + (Y) ND (Y) ND (Y) aquaglyceroporin, EcGlpF, is highly permeable to glycerol, but only very weakly permeable to urea [48]. Of the lactic-acidpermeable aqua(glycero)porins, rAQP9 is permeable to urea, whereas AtNIP2;1 is not [12]. In the case of the tested bacterial aquaglyceroporins, urea permeability was seen together with lactic acid permeability (LpGlpF1 and LpGlpF4), but not with glycerol permeability (LpGlpF4 and LpGlpF3). These data suggest that aquaglyceroporins that are permeable to urea are also permeable to lactic acid, whereas this is not the case for the only known lactic acid-transporting AQP AtNIP2;1 [12], supporting the notion that, although the size and chemical environment of the selectivity filter are important for the passage of urea, glycerol and lactic acid, there must be additional restriction sites for the passage of each substrate. A molecular dynamics simulation study [49] suggested that the pore environment next to the NPA filter region (towards the intracellular side) is a high-energy barrier for solutes such as urea. Examination of the protein sequences of the different isoforms (Supplementary Figure S1) did not indicate any specific amino acids that might be responsible for distinguishing between these three solutes. As expected, the H2 O2 sensitivity of yeasts expressing LpGlpF isoforms correlated closely with the measured water permeability. We also tried to identify key residues responsible for lactic acid permeability by comparing the sequences of previously described MIPs and the bacterial GlpF aquaglyceroporins. Three highly conserved residues, Gly64 , Ala65 and His66 , upstream of the first NPA motif, are known to interact with substrate molecules inside the channel of EcGlpF [49]. With the exception of Ala65 , which is replaced by a valine residue in all members of this group, these residues are much less well conserved in GlpF1– GlpF4 homologues, in which Gly64 is replaced by an asparagine, aspartate or glutamate residue and His66 by a cysteine, serine, phenylalanine or tyrosine residue, showing that these residues are of minor importance in lactic acid selectivity. The proline residue in the second NPA motif is also less well conserved in the GlpF1–GlpF4 homologues, being replaced by leucine or glutamine residue. However, replacement of the proline by other hydrophobic residues (leucine, methionine or valine) or polar uncharged residues (serine, threonine, asparagine or glutamine) is observed in the NPA motifs of MIPs from yeast, filamentous fungi, nematodes, frogs and sea urchins [50,51]. GlpF1–GlpF4 homologues and EcGlpF or GlpF2 homologues transport different molecules, but no difference was noted in the residues forming the ar/R selectivity filter (Supplementary Figure S1 and Table S5), indicating that these residues are not responsible for the lactic acid selectivity. It will be interesting to determine which residues account for the lactic acid transport of GlpF1–GlpF4 homologues. The Val71 residue located just downstream of the first NPA motif c The Authors Journal compilation c 2013 Biochemical Society is replaced by methionine in all GlpF1-4 homologues and in two GlpF5 homologues found in lar operons. This residue may be a hallmark of bacterial lactic acid transporters. However, in rAQP9, AtNIP2;1 and SmAQP, this residue is a valine. The existence of a glpF gene coding for an aquaglyceroporin transporting lactic acid in the lar operon of lactic acid bacteria is of particular interest. To the best of our knowledge, no lactic acid channel has been functionally characterized in lactic acid bacteria, so GlpF1 and GlpF4 represent the first proteins that contribute, in addition to the intrinsic membrane permeability, to lactic acid transport in these bacteria. In Xenopus oocytes, both lactic acid isomers were transported equally well by LpGlpF4, demonstrating that this channel does not favour the transport of one isomer over the other. Deletion of both glpF1 and glpF4 in Lb. plantarum WCFS1 demonstrated the involvement of these channels in the cellular racemization of lactic acid, justifying the presence of a lactic acid transporter in many lar operons from various Lactobacillales genomes. However, the location outside the lar operon of GlpF1 homologues, such as GlpF4 from Lb. plantarum, suggests a more general physiological role for these channels than only lactic acid racemization. Indeed, the role of GlpF-mediated lactic acid transport seems to be important during exponential growth phase, as shown by the competition experiment between the wild-type and the double mutant. Growth experiments also suggested that the uptake of lactic acid at the onset of stationary phase via LpGlpF1 and LpGlpF4 was responsible for the increased sensitivity to the extracellular non-dissociated form and provided further evidence that GlpFs function as lactic acid channels. Furthermore, our growth analyses of the different bacterial strains at different pH values suggested that lactic acid is channelled, but the lactate anion is not. The ability of MIPs to conduct lactic acid seems to be a conserved and common feature of the GlpF1/F4 subgroup. This was supported by the observation that PpGlpF1 and LsGlpF1, which are located in the lar operon, have the same substrate specificity as their Lb. plantarum homologues. The transport ability of GlpF isoforms belonging to the GlpF1/F4 group might be the basis for the protein-mediated facilitated diffusion of the non-dissociated lactic acid molecule, which was first suggested a long time ago, based on translocation specificity and kinetic analyses in several Lactobacillales species [52,53]. Whether aquaglyceroporins from Lb. plantarum are the only transporters responsible for the regulation of the transmembrane transport of lactic acid remains to be elucidated. The Lb. plantarum genome encodes a potential lactate permease protein (lp_1814) belonging to the lactate permease protein family, known as a lactate/H + symporter, and its E. coli homologue is involved Aquaglyceroporins from Lb. plantarum in uptake of lactate into bacteria [54]. Whether these two types of transporter are responsible for lactic acid transport under different physiological conditions remains to be elucidated. AUTHOR CONTRIBUTION Gerd Bienert and Benoı̂t Desguin performed the experimental work. Gerd Bienert, Benoit Desguin, François Chaumont and Pascal Hols designed the experiments, analysed the data and wrote the paper. FUNDING This work was supported by grants from the Belgian National Fund for Scientific Research (FNRS), the Interuniversity Attraction Poles Programme-Belgian Science Policy and the “Communauté Française de Belgique-Actions de Recherches Concertées”. G.P.B. was an FNRS Postdoctoral Researcher. B.D. and P.H. are a Research Fellow and Research Associate at the FNRS respectively. REFERENCES 1 Engel, A. and Stahlberg, H. (2002) Aquaglyceroporins: channel proteins with a conserved core, multiple functions, and variable surfaces. Int. Rev. Cytol. 215, 75–104 2 Ishibashi, K. (2009) New members of mammalian aquaporins: AQP10–AQP12. Handb. Exp. Pharmacol. 190, 251–262 3 Zardoya, R. (2005) Phylogeny and evolution of the major intrinsic protein family. Biol. 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BIENERT*1,2 , Benoı̂t DESGUIN*1 , François CHAUMONT*3 and Pascal HOLS*3 *Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 4-5, 1348 Louvain-la-Neuve, Belgium Table S1 Strains and plasmids used in the present study r Cm , resistance to chloramphenicol; Emr , resistance to erythromycin; NCIMB, The National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland; StrR , resistance to streptomycin. Strain or plasmid Strain S. cerevisiae YNVW1 Lb. plantarum WCFS1 LR0003 LR0004 LR0005 E. coli TOP10 Plasmid pNB1u pNB1YFPu pRS426-pTPIu pNZ5319 pNZ5348 pGIR801 pGIR802 1 2 3 Characteristic(s) Source or reference dur3 , ura3 ; mutant defective in urea uptake [1] Wild-type WCFS1 glpF1 WCFS1 glpF4 WCFS1 glpF1 ΔglpF4 NCIMB The present study The present study The present study F − mcrA (mrr-hsdRMS-mcrBC ) ϕ80 lacZ M15 lacX 74 nupG recA1 araD 139 (ara-leu)7697 galE 15 galK 16 rpsL (StrR ) endA1 λ − Invitrogen Oocyte expression vector for MIP homologues Oocyte expression vector for the N-terminal YFP-tagged protein of interest fusion Yeast expression vector for MIP homologues Cmr Emr ; pNZ5318 derivative for multiple gene replacements in Gram-positive bacteria Emr ; pGID023 derivative containing cre under the control of the lp_1144 promoter Cmr Emr ; pNZ5319 derivative containing homologous regions up- and down-stream of glpF1 (used for the construction of LR0003) Cmr Emr ; pNZ5319 derivative containing homologous regions up- and down-stream of glpF4 (used for the construction of LR0004) [2] [2] Laboratory collection [3] [3] The present study The present study These authors contributed equally to this work. Present address: Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany. Correspondence may be addressed to either of these authors (email [email protected] or [email protected]). c The Authors Journal compilation c 2013 Biochemical Society G. P. Bienert and others Figure S1 ClustalX2 alignment of the six MIPs from Lb. plantarum (LpGlpF1–LpGlpF6), a Schistosoma mansoni AQP (SmAQP), Rattus norvegicus AQP9 (rAQP9), E. coli GlpF and AqpZ (EcGlpF and EcAqpZ), and Arabidopsis thaliana NIP2-1 (AtNIP2-1) The selectivity ar/R filter residues (H2, H5, LE1 and LE2 ) and the NPA motifs (NPA1 in loop B and NPA2 in loop E) are boxed (see Table S5 for details). c The Authors Journal compilation c 2013 Biochemical Society Aquaglyceroporins from Lb. plantarum Figure S2 Confocal microscopy analysis of YFP-tagged GlpF proteins expressed in X. laevis oocytes Oocyte injected with 25 ng of cRNA coding for YFP–LpGlpF1 (A), YFP–LpGlpF2 (B), YFP–LpGlpF3 (C), YFP–LpGlpF4 (D), YFP–LpGlpF5 (E) or YFP–LpGlpF6 (F), or with 4 ng of cRNA coding for YFP–ZmPIP2;5 (G) or 50 nl of water (H) were fixed and observed 3 days after injection as described previously [9]. The arrows indicate labelling of the oocyte membrane. Scale bars, 100 μm. Figure S3 Water permeability of X. laevis oocytes expressing Lb. plantarum GlpFs (A) Osmotic water permeability coefficients (P f ) of oocytes injected with water (negative control) or cRNA coding for ZmPIP2;5 or co-injected with cRNAs coding for ZmPIP2;5 and LpGlpF1, LpGlpF5 or LpGlpF6. The data are expressed as the means of measurements of 8–13 cells. (B) P f values for oocytes injected with water (negative control) or cRNA coding for rAQP9, ZmPIP2;5, LpGlpF2, LpGlpF3 or LpGlpF4. The results are expressed as the means of measurements of 6–9 cells. In both experiments, 25 ng of cRNA for each MIP-encoding gene was used, except in the case of ZmPIP2;5 (2 ng). The error bars are the 95 % confidence intervals. Figure S4 Lactic acid membrane permeability of X. laevis oocytes expressing Lb. plantarum GlpFs D/L-lactic acid membrane permeability of oocytes injected with water (negative control) or cRNA coding for rAQP9 (positive control), LpGlpF2, LpGlpF3 or LpGlpF4, or co-injected with cRNAs coding for ZmPIP2;5 and LpGlpF1, LpGlpF5 or LpGlpF6. The initial swelling rates were measured by immersion for 350 s in an isotonic modified Barth solution containing 80 mM D/L-lactate with the osmolarity adjusted to 200 mOsm/l with NaCl. V (t) indicates the volume at a given time point and V (0) the initial volume. The data are expressed as the mean of measurements of 8–12 oocytes in one representative experiment out of three. The error bars are the 95 % confidence intervals. c The Authors Journal compilation c 2013 Biochemical Society G. P. Bienert and others Table S2 List of primers used in the present study (a) Primers for USER-compatible Xenopus gene expression Primer name Sequence (5 →3 ) Mutant strain generation or plasmid construction GlpF1_A GlpF1_B GlpF2_A GlpF2_B GlpF3_A GlpF3_B GlpF4_A GlpF4_B GlpF5_A GlpF5_B GlpF6_A GlpF6_B GlpSak_A GlpSak_B GlpPen_A GlpPen_B GGCTTAAUATGGTACATCAGTTGATTGCTGAATTTATGG GGTTTAAUTTAATTAATTCCAAAAAAGCCATGCATAAAC GGCTTAAUATGCACGGTTTTTTAGGTGAATTTTTAG GGTTTAAUTTAACTAATAATTGTTTCCAGACCAG GGCTTAAUATGATGAAAGATCCATTAGCACTAC GGTTTAAUTTATGGCAATACATTAAACAACAG GGCTTAAUATGATTCATCAGCTTTTGGCAGAG GGTTTAAUCTACCCAATCCCAAAGAACC GGCTTAAUATGACAGGTTCATGGGAAG GGTTTAAUTTATAAACCAAAAAAGATCTTATAAATCC GGCTTAAUATGCGCAAGTATCTCGCC GGTTTAAUCTAGTCTTCTGATCCCATAACC GGCTTAAUATGGTTCATCAGTTATTAGCAGAATTCATGG GGTTTAAUTTAGATACCAAAGAAGCTCCGCATGAAG GGCTTAAUATGGTAAATCATCTATTAGCCGAATTTATGG GGTTTAAUTTAGTAAATGTGTAAGTAAAATCTAACAAAAG LpGlpF1 expression LpGlpF1 expression LpGlpF2 expression LpGlpF2 expression LpGlpF3 expression LpGlpF3 expression LpGlpF4 expression LpGlpF4 expression LpGlpF5 expression LpGlpF5 expression LpGlpF6 expression LpGlpF6 expression LsGlpF1 expression LsGlpF1 expression PpGlpF1 expression PpGlpF1 expression (b) Primers used for the construction of deletion vectors Primer name Sequence (5 →3 ) Mutant strain generation or plasmid construction -D_UP_A -D_UP_B -D_DW_A -D_DW_B -GlpF4_UP_A -GlpF4_UP_B -GlpF4_DW_A -GlpF4_DW_B TGATATGTTTCTGGGCGCGTTAC TACCAAAAAATTACGCCTCCTCATC TGCCAGGAATTGCGCCCTTTG TTGCACCGTGACCAACTTGTAAC GAATTTAAAAGCCCGTTACCC AAGCTGATGAATCACTATCTTTCTC ATGGGTTCTTTGGGATTGGGTAG TCACGTTGCCCTCCTGCTTTG pGIR801 pGIR801 pGIR801 pGIR801 pGIR802 pGIR802 pGIR802 pGIR802 Primer name Sequence (5 →3 ) Mutant strain generation or plasmid construction 2Hy_LarC_A -D_diag_A -GlpF4_diag_A -GlpF4_diag_B GATGCAAACACTTTATTTAGACGCTTTTTC GCCGCCTTATTAGCTTTGG GTTTCCCGCGGCATTCCATTG TTGGTGCATATCATAGTACCGATATTG LR0003 LR0003 LR0004 LR0004 (c) Primers used for the validation of deletions (d) Primers used for the sequencing of deletion vectors Primer name Sequence (5 →3 ) Mutant strain generation or plasmid construction 85 85_compl 87_bis 87_bis_compl GTTTTTTTCTAGTCCAAGCTCACA TTATTCGTTTGATTTCGCTTTCG TTGATGATTGGTTCGGAAGGCACG TATATAGTTTACCCCGTCAGC pGIR801 and pGIR802 pGIR801 and pGIR802 pGIR801 and pGIR802 pGIR801 and pGIR802 c The Authors Journal compilation c 2013 Biochemical Society Aquaglyceroporins from Lb. plantarum Table S3 Genetic context of Lb. plantarum glpFs (a) glpF1 gene context Accession number Protein Annotation YP_004888174.1 YP_004888175.1 YP_004888176.1 YP_004888177.1 YP_004888178.1 YP_004888179.1 larA gene product larB gene product larC1 gene product larC2 gene product glpF1 gene product larE gene product Lactate racemization operon protein LarA Lactate racemization operon protein LarB Lactate racemization operon protein LarC, N-terminal domain Lactate racemization operon protein LarC, C-terminal domain Glycerol uptake facilitator protein GlpF1 Lactate racemization operon protein LarE (b) glpF2 gene context Accession number Protein Annotation YP_004888225.1 YP_004888226.1 YP_004888227.1 YP_004888228.1 YP_004888229.1 dak1B gene product dak2 gene product dak3 gene product dhaP gene product PurR Phosphotransferase, dihydroxyacetone-binding subunit Dihydroxyacetone phosphotransferase, ADP-binding subunit Dihydroxyacetone phosphotransferase, phosphoryl donor protein Dihydroxyacetone transport protein GlpF2 LacI family transcriptional regulator, maltose-related (c) glpF3 gene context Accession number Protein Annotation YP_004888401.1 YP_004888402.1 YP_004888403.1 glpK gene product glpD gene product glpF3 gene product Glycerol kinase Glycerol-3-phosphate dehydrogenase, FAD-dependent Glycerol uptake facilitator protein GlpF3 (d) glpF4 gene context Accession number Protein Annotation YP_004889075.1 YP_004889076.1 YP_004889077.1 Virul_fac_BrkB glpF4 gene product glf1 gene product Ribonuclease BN family protein Glycerol uptake facilitator protein GlpF4 UDP-galactopyranose mutase (e) glpF5 gene context Accession number Protein Annotation YP_004890928.1 YP_004890929.1 YP_004890930.1 YP_004890931.1 cadA gene product glpF5 gene product trxA3 gene product BsYrkD-like Cadmium transporting P-type ATPase Glycerol uptake facilitator protein GlpF5 Thioredoxin Hypothetical protein Accession number Protein Annotation YP_004890947.1 YP_004890948.1 YP_004890949.1 MoxR glpF6 gene product ubiB gene product MoxR family ATPase Glycerol uptake facilitator protein GlpF6 Ubiquinone biosynthesis protein, ABC1 family (e) glpF6 gene context c The Authors Journal compilation c 2013 Biochemical Society G. P. Bienert and others Table S4 List of 49 MIPs from 17 Lactobacillales species The number in front of the MIP name corresponds to that after the species name in Figure 1 of the main text and Figure S1. Dha, dihydroxyacetone utilization operon [4]; Gly, glycerol utilization operon [5]; Lar, lactate racemization operon [6]; and Prop, propanediol utilization operon [7]. Species S. agalactiae FSL S3-025 1 2 3 Weissella koreensis KACC 15509 1 2 Enterococcus faecalis OG1RF 1 2 3 Leuconostoc mesenteroides subsp. mesenteroides J17 1 2 Lactobacillus acidophilus 30SC 1 Lb. salivarius SMXD51 1 2 3 Lactobacillus suebicus KCTC 3549 1 2 3 4 Lactobacillus mali KCTC 3596 = DSM 20444 1 2 3 Lc. lactis subsp. cremoris NZ9000 1 2 3 Leuconostoc argentinum KCTC 3773 1 2 Lactobacillus delbrueckii subsp. lactis DSM 20072 1 2 Streptococcus australis A.T.C.C. 700641 1 2 3 4 Granulicatella adiacens A.T.C.C. 49175 1 Lb. plantarum WCFS1 1 2 3 4 5 6 Lb. brevis ATCC 367 1 2 3 P. pentosaceus ATCC 25745 1 2 3 Lb. sakei subsp. sakei 23K 1 2 3 4 c The Authors Journal compilation c 2013 Biochemical Society Accession number Annotation Context EGS28598.1 EGS26706.1 EGS26627.1 Glycerol uptake facilitator protein Hypothetical protein FSLSAGS3026_10000 Glycerol uptake facilitator protein Gly Dha YP_004726868.1 YP_004726362.1 MIP family facilitator protein Aquaporin - YP_005708647.1 YP_005708605.1 YP_005708021.1 glpF2 gene product glpF gene product aqpZ gene product Gly Lar - YP_005174634.1 YP_005173456.1 Glycerol uptake facilitator-related permease (MIP family) Glycerol uptake facilitator-related permease - YP_004292761.1 Glycerol uptake facilitator protein - EIA33094.1 EIA32770.1 EIA32549.1 Glycerol uptake facilitator protein Glycerol uptake facilitator protein Glycerol uptake facilitator protein Dha Gly ZP_09451087.1 ZP_09450612.1 ZP_09449835.1 ZP_09449755.1 Glycerol uptake facilitator-related permease Glycerol uptake facilitator protein MIP family glycerol uptake facilitator protein GlpF Glycerol uptake facilitator-related permease (MIP family) - ZP_09449322.1 ZP_09448664.1 ZP_09448262.1 Glycerol uptake facilitator protein Glycerol uptake facilitator protein Glycerol uptake facilitator-related permease (MIP family) Dha Lar YP_006357717.1 YP_006356524.1 YP_006356304.1 Putative glycerol uptake facilitator protein Glycerol uptake facilitator Transporter Gly - ZP_08230752.1 ZP_08230396.1 Glycerol uptake facilitator-related permease (MIP family) Aquaporin Z - EGD27475.1 EGD27267.1 MIP family glycerol uptake facilitator protein GlpF MIP family glycerol uptake facilitator protein GlpF - ZP_08021527.1 ZP_08021095.1 ZP_08021080.1 ZP_08020846.1 Glycerol facilitator-aquaporin MIP family glycerol uptake facilitator protein GlpF MIP family glycerol uptake facilitator protein GlpF MIP family major intrinsic protein water channel AqpZ Prop Gly - ZP_05738516.1 Glycerol uptake facilitator Lar YP_004888178.1 YP_004888228.1 YP_004888403.1 YP_004889076.1 YP_004890929.1 YP_004890948.1 glpF1 gene product dhaP gene product (GlpF2) glpF3 gene product glpF4 gene product glpF5 gene product glpF6 gene product Lar Dha Gly - YP_795947.1 YP_795727.1 YP_796082.1 Glycerol uptake facilitator-related permease (MIP family) Glycerol uptake facilitator-related permease (MIP family) Glycerol uptake facilitator-related permease Lar - YP_805154.1 YP_805088.1 YP_804732.1 Glycerol uptake facilitator-related permease (MIP family) Glycerol uptake facilitator-related permease (MIP family) Glycerol uptake facilitator-related permease Lar Gly - YP_395402.1 YP_395266.1 YP_395780.1 YP_396456.1 MIP family facilitator protein glpF gene product MIP family facilitator protein aqpZ gene product Lar Gly - Aquaglyceroporins from Lb. plantarum Table S5 Selectivity filter residues and NPA boxes in Lb. plantarum , Lb. sakei and P. pentosaceus MIPs The ar/R filter is defined by one amino acid residue in helix 2 (H2), one in helix 5 (H5) and two in loop E (LE). The ar/R region is the narrowest part of the pore and the electro-physicochemical properties of the four amino acids forming this selectivity filter are assumed to determine the pore selectivity of MIPs in general. As in EcGlpF, this selectivity filter in GlpF1 and GlpF2 is composed of WGFR (see also Figure S1), GlpF3 has a WGYR ar/R selectivity filter, which is physicochemically very similar to WGFR, GlpF5 a rather unusual YVPR ar/R filter and GlpF6 (see also Figure S1) F(H/I)XR, in which the X residue is a small uncharged residue (e.g. A, G, L or T), which is typical of the water-specific AQP family in several organisms [8]. The amino acids corresponding to the characteristic MIP NPA motifs in loop B and E are shown on the right-hand side; changes from the classical NPA motifs are shaded in grey. ar/R selectivity filter NPA motifs Protein H2 H5 LE1 LE2 LoopB LoopE LpGlpF1 LpGlpF2 LpGlpF3 LpGlpF4 LpGlpF5 LpGlpF6 LsGlpF1 PpGlpF1 W W W W Y F W W G G G G V I G G F F Y F P G F F R R R R R R R R NPA NPA NPA NPA NPA NPA NPA NPA NLA NPA NPA NLA NPA NPA NLA NQR REFERENCES 1 Liu, L. H., Ludewig, U., Frommer, W. B. and von Wiren, N. (2003) AtDUR3 encodes a new type of high-affinity urea/H + symporter in Arabidopsis . Plant Cell 15, 790–800 2 Nour-Eldin, H. H., Hansen, B. G., Norholm, M. H., Jensen, J. K. and Halkier, B. A. (2006) Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res. 34, e122 3 Lambert, J. M., Bongers, R. S. and Kleerebezem, M. (2007) Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum . Appl. Environ. Microbiol. 73, 1126–1135 4 Zurbriggen, A., Jeckelmann, J. M., Christen, S., Bieniossek, C., Baumann, U. and Erni, B. (2008) X-ray structures of the three Lactococcus lactis dihydroxyacetone kinase subunits and of a transient intersubunit complex. J. Biol. Chem. 283, 35789–35796 5 Durnin, G., Clomburg, J., Yeates, Z., Alvarez, P. J., Zygourakis, K., Campbell, P. and Gonzalez, R. (2009) Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli . Biotechnol. Bioeng. 103, 148–161 6 Goffin, P., Deghorain, M., Mainardi, J. L., Tytgat, I., Champomier-Verges, M. C., Kleerebezem, M. and Hols, P. (2005) Lactate racemization as a rescue pathway for supplying D-lactate to the cell wall biosynthesis machinery in Lactobacillus plantarum . J. Bacteriol. 187, 6750–6761 7 Johnson, C. L., Pechonick, E., Park, S. D., Havemann, G. D., Leal, N. A. and Bobik, T. A. (2001) Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene. J. Bacteriol. 183, 1577–1584 8 Savage, D. F., O’Connell, 3rd, J. D., Miercke, L. J., Finer-Moore, J. and Stroud, R. M. (2010) Structural context shapes the aquaporin selectivity filter. Proc. Natl. Acad. Sci. U.S.A. 107, 17164–17169 9 Bienert, G. P., Cavez, D., Besserer, A., Berny, M. C., Gilis, D., Rooman, M. and Chaumont, F. (2012) A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers. Biochem. J. 445, 101–111 Received 14 March 2013/20 June 2013; accepted 25 June 2013 Published as BJ Immediate Publication 25 June 2013, doi:10.1042/BJ20130388 c The Authors Journal compilation c 2013 Biochemical Society