Maria Veiga-Da-Cunha

Tatiana Sokolova

Fred Opperdoes

Emile Van Schaftingen
email: emile.vanschaftingen@uclouvain.be

Evolution of vertebrate glucokinase regulatory protein from a bacterial Nacetylmuramate 6-phosphate etherase

Keywords: muramyl-peptide, N-acetylmuramate, fructose metabolism, GKRP, fructose 6phosphate, N-acetylmurate 6-phosphate GK, glucokinase, hiGK, human islet glucokinase, xGK, xenopus glucokinase, GKRP, glucokinase regulatory protein, rGKRP, rat glucokinase regulatory protein, xGKRP, xenopus glucokinase regulatory protein, MurNAc6P

Mammalian glucokinase regulatory protein (GKRP), a fructose 6-phosphate and fructose 1phosphate sensitive inhibitor of glucokinase (GK), appears to have resulted from the duplication of a gene similar to bacterial N-acetylmuramate 6-phosphate etherase (MurQ). In the present work, we show that several genomes of primitive eukaryotes encode a GKRP-like protein with two MurQ repeats. Recombinant Haemophilus influenzae MurQ and the GKRP homologue of the amoebaflagellate Naegleria gruberi both behaved as excellent Nacetylmuramate 6-phosphate etherases, with K cat values (83 and 20 sec -1 ) at least as high as that reported for Escherichia coli MurQ. By contrast, rat and xenopus GKRP displayed much lower etherase activities (K cat = 0.08 and 0.05 sec -1 , respectively). The etherase activity of rat GKRP was inhibited by ligands (fructose 6-phosphate, fructose 1-phosphate, sorbitol 6phosphate) known to regulate its interaction with GK and by mutations affecting the binding of these phosphate esters. This indicated that these phosphate esters all bind to a single regulatory site, which evolved from the original catalytic site. Sorbitol 6-phosphate and other phosphate esters also inhibited the etherase activity of xenopus GKRP, but did not affect its ability to inhibit GK. Thus, unlike what was previously thought, xenopus GKRP has a binding site for phosphate esters, but this site is uncoupled from the GK binding site. Taken together, these data indicate that duplication of the murQ gene led to a eukaryotic type etherase, which subsequently evolved to GKRP by acquiring a new binding specificity whilst losing most of its etherase activity.

INTRODUCTION

Studies on the effect of fructose to stimulate the phosphorylation of glucose by glucokinase (GK) in rat hepatocytes led to the identification of glucokinase regulatory protein (GKRP) [START_REF] Van Schaftingen | Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes[END_REF][START_REF] Van Schaftingen | A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate[END_REF]. This 68 kDa protein binds and inhibits GK, and its effect is reinforced by fructose 6phosphate and antagonized by fructose 1-phosphate [START_REF] Vandercammen | The mechanism by which rat liver glucokinase is inhibited by the regulatory protein[END_REF]. Fructose 1-phosphate, which is formed from fructose by ketohexokinase, acts at concentrations in the micromolar range and accounts for the stimulatory effect that low concentrations of fructose have on the phosphorylation of glucose. Similar regulatory properties are observed with human GKRP [START_REF] Brocklehurst | Differences in regulatory properties between human and rat glucokinase regulatory protein[END_REF]. GKRP is also involved in the control of the subcellular localization of GK, which is present in the nucleus at low concentrations of glucose, but moves to the cytosol when the concentration of glucose is elevated or if low concentrations of fructose are present [START_REF] Agius | Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin[END_REF][START_REF] De La Iglesia | Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase[END_REF]. Mice deficient in GKRP have reduced GK activity and this enzyme is exclusively cytosolic in their hepatocytes, suggesting that GKRP serves also to maintain in the nucleus a functional reserve of GK that is quickly available in the presence of glucose [START_REF] Farrelly | Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: a sequestration mechanism in metabolic regulation[END_REF][START_REF] Grimsby | Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels[END_REF]. Recently, two polymorphisms in the GKRP gene were shown to be linked with the blood glucose and triglyceride levels in various human populations [9,[START_REF] Orho-Melander | Common missense variant in the glucokinase regulatory protein gene is associated with increased plasma triglyceride and C-reactive protein but lower fasting glucose concentrations[END_REF][START_REF] Vaxillaire | The common P446L polymorphism in GCKR inversely modulates fasting glucose and triglyceride levels and reduces type 2 diabetes risk in the DESIR prospective general French population[END_REF][START_REF] Qi | Association of GCKR rs780094, alone or in combination with GCK rs1799884, with type 2 diabetes and related traits in a Han Chinese population[END_REF], emphasizing the importance of GKRP in the control of carbohydrate metabolism, but also suggesting a link between this regulatory molecule and lipid metabolism.

Detailed characterization of rat GKRP showed that several sugar or polyol-phosphates, most particularly sorbitol 6-phosphate, mimic the effect of fructose 6-phosphate, whereas others mimic the effect of fructose 1-phosphate [START_REF] Detheux | Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation[END_REF]. Several residues that are involved in the binding of these phosphate esters were identified by site-directed mutagenesis [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF][START_REF] Veiga-Da-Cunha | Mutations in the glucokinase regulatory protein gene in 2p23 in obese French caucasians[END_REF]. Intriguingly, xenopus GKRP, which shares about 60 % sequence identity with its mammalian homologues, inhibits GK in a fructose 6-phosphate and fructose 1-phosphate insensitive manner [START_REF] Veiga-Da-Cunha | Cloning and expression of a Xenopus liver cDNA encoding a fructose-phosphate-insensitive regulatory protein of glucokinase[END_REF].

Sequence comparisons indicate that GKRP belongs to the sugar isomerase (SIS) family of proteins [START_REF] Bateman | The SIS domain: a phosphosugar-binding domain[END_REF]. Its closest homologues are bacterial proteins (MurQ) involved in the recycling of the peptidoglycan and catalysing the hydrolysis of the ether bond in Nacetylmuramate 6-phosphate, thus forming N-acetylglucosamine 6-phosphate and D-lactate, [18,[START_REF] Uehara | MurQ Etherase is required by Escherichia coli in order to metabolize anhydro-N-acetylmuramic acid obtained either from the environment or from its own cell wall[END_REF][START_REF] Hadi | Mechanistic studies on Nacetylmuramic acid 6-phosphate hydrolase (MurQ): an etherase involved in peptidoglycan recycling[END_REF]. The GKRP sequence ( 600 residues), which has about twice the size of the MurQ sequence ( 300 residues), contains two regions that are homologous to MurQ. As MurQ is active as a homodimer [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF], whereas GKRP is a monomer (it heterodimerizes with GK), it has been suggested that the GKRP structure resembles that of a MurQ dimer [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF][START_REF] Kim | Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling[END_REF].

An interesting question is that of the evolution of GKRP, most particularly since there is no obvious connection between muramyl-peptide recycling in bacteria and the allosteric regulation of an enzyme (vertebrate GK) that appeared late in evolution. Are there GKRP homologues in primitive eukaryotes and what is their function? Based on sequence comparisons, Jaeger and Mayer [START_REF] Jaeger | The transcriptional factors MurR and catabolite activator protein regulate N-acetylmuramic acid catabolism in Escherichia coli[END_REF] proposed that the gene duplication event that led to present day GKRP pre-dates the separation of bacterial etherases from vertebrate GKRP. One may therefore wonder if the ancestral gene that underwent this duplication event encoded a protein with etherase activity or with another function. The purpose of the present paper was to shine some light on the evolution of GKRP.

EXPERIMENTAL Reagents

D-lactate dehydrogenase (from Lactobacillus Leichmannii) and pig heart glutamate-pyruvate transaminase were from Roche diagnostics (Mannheim, Germany). Glycylglycine, Lglutamate, N-acetylmuramic acid and N-acetyl-D-glucosamine 6-phosphate disodium salt were from Sigma-Aldrich (St. Louis, MO, USA). Molecular biology DNA modifying enzymes and Pfu polymerase were from Fermentas GMBH, Germany. Optimase polymerase was from Transgenomics, 78990 Elancourt, France. pET expression vectors were from Novagen, Darmstadt, Germany. Recombinant Haemophilus influenzae YfeU (or MurQ) [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF], xenopus liver GK and GKRP [START_REF] Veiga-Da-Cunha | Cloning and expression of a Xenopus liver cDNA encoding a fructose-phosphate-insensitive regulatory protein of glucokinase[END_REF] and human beta-cell GK [START_REF] Veiga-Da-Cunha | Effect of mutations on the sensitivity of human beta-cell glucokinase to liver regulatory protein[END_REF] were expressed in Escherichia coli and purified as previously described.

Phylogenetic analysis

The Naegleria gruberi sequence was placed into an alignment of 2 selected bacterial Nacetylmuramic acid 6-phosphate etherase and 15 eukaryotic GKRP homologues obtained from the NCBI Genebank and TrEMBL databases. A total of 235 unambiguously aligned sites common to all sequences (Supplementary Figure S1) was retained for phylogenetic analysis. Phylogenetic trees were inferred by the Neighbour-joining method [START_REF] Felsenstein | Evolutionary trees from DNA sequences: a maximum likelihood approach[END_REF] as implemented by the program ClustalX version 2 [START_REF] Thompson | The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF] and by Bayesian analysis using MrBAYES version 3.1.2 [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogeny[END_REF]. The BLOSUM model of evolution and a gamma rate distribution with 4 substitution rate categories fitted best the data. To estimate Bayesian posterior probabilities, Markov Chain Monte Carlo (MCMC) chains were run for 80,000 generations and sampled every 100 generations (burn-in: 20,000 generations). The final dataset for tree construction comprised the following taxa (accession numbers): N. gruberi (Naegrl 78308; http://genome.jgi-psf.org/Naegr1/Naegr1.home.html), Xenopus laevis (Q91754), Mus musculus (Q91X44), Rattus rattus (Q07071), Homo sapiens (Q14397), Canis familiaris (XP_532912.2), Danio rerio (XP_001334641.2), Tetraodon nigrovoridis (CAG06348.1), Nematostella vectensis (XP_001640085.1), Branchiostoma floridae (XP_002249995.1), Strongylocentrotus purpuratus (XP_001198229.1), Monosiga brevicolis (XP_001750465.1), Dictyostelium discoideum (XP_646964.1), Micromonas pusilla (EEH52070.1), Ciona intestinalis (XP_002123418.1). The sequences of E. coli (P76535) and H. influenzae (P44862) were used as outgroups.

Overexpression and purification of recombinant proteins

Recombinant proteins were expressed with a C-terminal (rat GKRP and N. gruberi GKRP homologue) or a N-terminal (N-acetylglucosamine kinase, N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase/isomerase) 6xHis tag. pBlueScript containing rat GKRP [START_REF] Detheux | Heterologous expression of an active rat regulatory protein of glucokinase[END_REF] was utilized to PCR amplify the C-terminal end ( 300 bp) of rat GKRP, using Pfu polymerase. This PCR-amplified product was used to replace the Cterminal end of rat GKRP in the original pBlueScript-GKRP plasmid. After sequencing confirmation, the NdeI/XhoI fragment containing the entire rat GKRP open reading frame without the stop codon ( 2000 bp) was excised from pBlueScript and ligated in the pET-24a expression vector. Since N. gruberi genes do not have introns, the open reading frame of the GKRP homologue was PCR amplified using genomic DNA from strain DRI using Optimase S1. Following sequence confirmation, the expression plasmids were introduced in E. coli BL21(DE3) pLyS. To produce the recombinant proteins, 500 ml of the respective bacterial cultures were grown in Luria-Bertani (LB)-rich medium and the proteins were expressed after addition of 0.4 mM isopropyl-D-thiogalactopyranoside for 18h at 16°C (rat GKRP and N. gruberi GKRP homologue) or for 5h (glucosamine 6-phosphate deaminase/isomerase and Nacetylglucosamine 6-phosphate deacetylase) or 18h (N-acetylglucosamine kinase) at 37°C.

Preparation of the bacterial extracts (25 ml) and purification of the 6xHis-tagged proteins on a 1-ml HisTrap column (Ni 2+ form; GE Healthcare) were done as described previously [28]. Protein elution with imidazole was confirmed by SDS-PAGE ( 68 kDa for GKRPs, 30 kDa for N-acetylglucosamine kinase, 39 kDa for N-acetylglucosamine 6phosphate deacetylase and 36 kDa for glucosamine 6-phosphate deaminase/isomerase). The selected fractions were concentrated on 15 ml Vivaspin ultrafiltration columns (Sartorius, Stockport, United Kingdom), and desalted on PD-10 columns (GE Healthcare) equilibrated with 25 mM Hepes, pH 7.4, 10 mM NaCl and 1 mM dithiothreitol. Protein concentration was estimated by measuring A 280 assuming molar absorptivities of 48,525 (rat GKRP), 39,600 (N. gruberi GKRP homologue), 28,210 (N-acetylglucosamine kinase), 18,700 (Nacetylglucosamine 6-phosphate deacetylase) and 12,740 M -1 .cm -1 (glucosamine 6-phosphate deaminase/isomerase). From 1 liter of culture we obtained 10, 18, 20, 40 or 8 mg of pure rat GKRP, N. gruberi GKRP homologue and S. pomeroyi N-acetylglucosamine kinase, Nacetylglucosamine 6-phosphate deacetylase or glucosamine 6-phosphate deaminase/isomerase, respectively. The purified proteins were supplemented with 10% glycerol and stored at -70 °C.

Synthesis of N-acetylmuramate 6-phosphate

N-acetylmuramate 6-phosphate was synthesized by incubating 10 mM N-acetylmuramate for 3 h at 37°C with purified recombinant N-acetylglucosamine kinase (70 g/ml) in the presence of 50 mM Hepes, pH 7.4, 15 mM ATP-Mg 2+ , 10 mM MgCl 2 , 1 mM dithiothreitol and 100 g/ml bovine serum albumin. To stop the reaction, the mixture was heated at 80°C for 10 min and centrifuged. The supernatant containing the resulting N-acetylmuramate 6-phosphate was purified by anion-exchange chromatography on a 15 ml Dowex 1-X8, 100-200 mesh resin equilibrated with 20 mM Hepes, pH 6.7. N-acetylmuramate 6-phosphate was eluted from the column using a linear NaCl gradient (0-1 M in 150 ml of buffer) and assayed as the inorganic phosphate [START_REF] Fiske | The colorimetric determination of phosphorus[END_REF] liberated by alkaline phosphatase. The preparation was stored at -20°C. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Assay for etherase activity

MurQ, xenopus and rat GKRPs etherase activities on N-acetylmuramate 6-phosphate were assayed through the formation of D-lactate or N-acetylglucosamine 6-phosphate. The recombinant proteins were incubated for 10 min at 30°C (H. influenzae MurQ), or 20 min (xenopus GKRP) or 40 min (rat GKRP) at 37°C in the reaction medium (0.12 ml) containing 25 mM Hepes pH7.4, 10 mM KCl, 1 mM MgCl 2 , 2 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, and the indicated concentrations of N-acetylmuramate 6-phosphate, in the presence or in the absence of different phosphoric esters. To stop the reaction, the mixture was heated for 5 min at 80°C and D-lactate was measured in the supernatant after centrifugation at 4°C for 10 min at 16,000 x g. The formation of D-lactate was measured spectophotometrically at 340 nm using an enzymatic assay based on the oxidation of Dlactate to pyruvate by the stereoisomer specific D-lactate dehydrogenase with reduction of NAD + to NADH+H + in the presence of L-glutamate and glutamate-pyruvate transaminase [START_REF] Gawehn | Lactate, in Methods of Enzymatic Analysis[END_REF]. The assay was done in a total volume of 0.6 ml of 200 mM glycylglycine-L-glutamate buffer, pH 10, containing 3.5 mM NAD + , 7 U of pig heart glutamate-pyruvate transaminase (Roche Applied Science) and 0.1 ml of sample. The absorbance at 340 nm was measured before the reaction was started by adding 15 U of L. leichmannii D-lactate dehydrogenase (Roche Applied Science). The change in absorbance at 340 nm, measured after 60 min at 37°C, corresponded to the amount of D-lactate produced during the delactoylation of Nacetylmuramate 6-phosphate.

N-acetylglucosamine 6-phosphate, the second product formed during the etherase reaction, was monitored using a NADP + coupled spectrophotometric assay at 340 nm. Nacetylglucosamine 6-phosphate was first deacetylated by N-acetylglucosamine 6-phosphate deacetylase and the product of the reaction, glucosamine 6-phosphate, was subsequently converted to fructose 6-phosphate by glucosamine 6-phosphate deaminase/isomerase. The fructose 6-phosphate produced was measured spectophotometrically in an assay mixture (0.6 ml) containing 25 mM Hepes pH 7.4, 10 mM KCl, 1 mM MgCl 2 , 2 mM dithiothreitol, 0.5 mM NADP + , 5 l of purified recombinant N-acetylglucosamine 6-phosphate deacetylase (1.2 g/ml), 3.5 U of yeast phosphoglucose isomerase, and 3.5 U of yeast glucose-6-phosphate dehydrogenase (Roche Applied Science). The reaction was started after 5 min preincubation at 30°C by addition of 15 l of purified recombinant glucosamine 6-phosphate deaminase/isomerase (2.2 g/ml) and the increase in A 340 was followed at 30°C. One unit of etherase activity is the amount of enzyme that converted 1 mol of N-acetylmuramate 6phosphate per min under the conditions described. K m and V max for the etherase activity of the various enzymes were calculated with the Prism 4 software, using the non-linear regression of hyperbola functionality.

Assays of GK and GKRP

GK was assayed at 30°C using a pyruvate kinase/lactate dehydrogenase-coupled assay [START_REF] Van Schaftingen | A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate[END_REF] with the exception that 1 mM DTT was also added. GKRP was assayed in the same assay by its ability to inhibit GK in the presence of 5 (human beta-cell GK) or 3 (xenopus GK) mM glucose and the indicated concentrations of phosphate esters. BLAST searches indicated that GKRP homologues with two MurQ repeats and a sequence length of 550-650 residues are found not only in mammals and amphibians, but also in fishes (D. rerio, T. nigroviridis), more primitive chordates (B. floridae, C. intestinalis), invertebrates (S. purpuratus, N. vectensis), and protists (M. brevicollis, D. discoideum, N. gruberi). No GKRP homologue with a duplicated MurQ sequence was found in prokaryotes.

RESULTS

Presence of GKRP homologues in primitive eukaryotes

It should be noted that fungi also have MurQ homologues with a larger (650-700 residues) size than bacterial MurQ. These putative proteins have a single MurQ repeat fused to a C-terminal domain homologous to N-acetylglucosamine kinase, suggesting that they are bifunctional proteins catalysing both the phosphorylation of N-acetylmuramate to Nacetylmuramate 6-phosphate and the hydrolysis of the ether bond in the latter. They were not included in our further analyses.

Sequence comparisons and structural considerations

GKRP from rat and xenopus and their homologue from N. gruberi are aligned with H. influenzae MurQ in Fig. 1. As previously noted for GKRPs, but now extended to more primitive GKRP homologues, the first 270 residues of these proteins align with residues 1-222 of MurQ, and the last 300 with residues 25 until the C-terminus of MurQ. In other words, the last 80 residues are missing in the first MurQ repeat of eukaryotic proteins. Consistent again with what had been observed with GKRPs [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF], the residues that are strictly conserved when comparing MurQ with the N-terminal part of the eukaryotic protein (underlined twice in the second MurQ repeat in Fig. 1) are most often (but not always) different from those that are conserved in the C-terminal part of the eukaryotic proteins and reciprocally. This applies also to GKRP homologues from more primitive eukaryotes.

Phylogenetic analysis (Fig. 2) based on the amino-acid residues shared by GKRP and MurQ homologues placed the bacterial N-acetylmuramate 6-phosphate etherase sequences and the eukaryotic two-domain etherase/GKRP sequences into two separate clades. The long branch of C. intestinalis must be an artefact because of the presence of some dubious residues in its sequence. All taxa within the eukaryotic clade follow the expected lines of descent, which suggest that the GKRP was not acquired late in evolution by an event of horizontal transfer from a bacterium, but that most likely a primitive eukaryotic ancestor already acquired a two-domain enzyme.

This evolution of GKRP can be understood with the help of the three-dimensional structure of H. influenzae MurQ (YfeU), a bacterial type N-acetylmuramate 6-phosphate etherase [START_REF] Kim | Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling[END_REF]. As previously proposed [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF][START_REF] Kim | Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling[END_REF][START_REF] Jaeger | The transcriptional factors MurR and catabolite activator protein regulate N-acetylmuramic acid catabolism in Escherichia coli[END_REF], functional MurQ is a dimer in which two identical subunits assemble to form two catalytic sites at their interface. Each of the catalytic sites is therefore made of residues from the two subunits. Fig. 3 shows the structure of the H. influenzae MurQ monomer [START_REF] Kim | Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling[END_REF], in which we have emphasized the residues that are strictly conserved in the amino-terminal part of rat GKRP (Fig. 3.A, residues in green and yellow) or in its carboxy-terminal part (Fig. 3.B, residues in blue and yellow). The yellow residues in both Fig. 3.A and 3.B correspond to those that are conserved in both regions. Strikingly, the residues conserved in the N-terminal "half" of GKRP form a cluster (Fig. 3.A) that is spatially different from the cluster formed by the residues that are conserved in the C-terminal "half" of GKRP (Fig. 3.B). Since both clusters are on the same face of the monomer and assuming that the subunits arrange in the MurQ dimer along a two-fold symmetry axis (yellow arrow in Fig. 3), it appears that only one of the two sites of the dimeric bacterial MurQ has been conserved in GKRPs. Thus, GKRP has a "pseudodimeric" structure, consisting of a first incomplete MurQ repeat linked to a second, nearly complete repeat, through a hinge region that is distal to the conserved catalytic site.

Etherase activity of H. influenzae MurQ, rat and xenopus GKRP and their N. gruberi homologue.

Rat GKRP and its N. gruberi homologue were produced with a 6xHis tag added to the Cterminal end of the protein. Xenopus GKRP [START_REF] Veiga-Da-Cunha | Cloning and expression of a Xenopus liver cDNA encoding a fructose-phosphate-insensitive regulatory protein of glucokinase[END_REF] and the H. influenzae MurQ [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF] were produced as unmodified proteins. All four recombinant proteins had the expected size, as determined by SDS-PAGE analysis and were purified to near-homogeneity. Their ability to catalyze the delactoylation of N-acetylmuramate 6-phosphate was determined by measuring the production of D-lactate and N-acetylglucosamine 6-phosphate, which were both assayed spectrophotometrically. As shown in Table 1, H. influenzae MurQ and the N. gruberi GKRP homologue showed substantial etherase activities, with comparable catalytic efficiencies. By contrast, rat and xenopus GKRP showed only a low, yet significant delactoylase activity, with K cat values more than two orders of magnitude lower than those of the bacterial or amoebal enzymes. Rat GKRP produced only about half as much N-acetylglucosamine 6-phosphate as D-lactate, whereas the three other proteins converted N-acetylmuramate 6-phosphate to stoichiometric amounts of the two products (not shown). We presume that rat GKRP also produced the alpha-beta unsaturated aldehyde form of N-acetylglucosamine 6-phosphate, a catalytic intermediate of the etherase reaction that is slowly released from the E. coli MurQ catalytic site [START_REF] Hadi | Mechanistic studies on Nacetylmuramic acid 6-phosphate hydrolase (MurQ): an etherase involved in peptidoglycan recycling[END_REF].

Modulation of the etherase activity by sugar-and polyol-phosphates

As mentioned in the Introduction, several phosphate esters bind to GKRP and modulate its interaction with GK [START_REF] Detheux | Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation[END_REF]. Their binding site might correspond to the catalytic site of GKRP and we therefore tested their effect on the etherase activity of rat GKRP and its three recombinant homologues. All tested effectors of rat GKRP inhibited the etherase activity of this protein (Fig. 4.A and 4.B) and their effects were consistent with what is known on their relative affinities for rat GKRP based on studies of its interaction with GK [START_REF] Detheux | Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation[END_REF]. Thus, sorbitol 6-phosphate (I 50 = 30 M, calculated from plots of 1/v versus [I]) was a more potent inhibitor of N-acetylmuramate 6-phosphate delactoylation than fructose 6-phosphate (I 50 = 130 M) (Fig. 4.A), another ligand for rat GKRP that reinforces GK inhibition. Among the compounds that release the inhibition exerted by rat GKRP on GK, the most potent etherase inhibitor was fructose 1-phosphate, followed by ribitol 5-phosphate and mannose 6-phosphate and finally ribose 5-phosphate (I 50 = 25, 80, 250 M, respectively) (Fig. 4.B). Glucose 6-phosphate, which does not affect the interaction between GK and rat GKRP, did not significantly inhibit the etherase activity, whereas N-acetylglucosamine 6-phosphate, the product of the reaction, exerted a modest inhibition (I 50 = 1 mM; Fig. 4.A).

The interaction of xenopus GKRP with GK is unaffected by fructose 6-phosphate and fructose 1-phosphate [START_REF] Veiga-Da-Cunha | Cloning and expression of a Xenopus liver cDNA encoding a fructose-phosphate-insensitive regulatory protein of glucokinase[END_REF][START_REF] Vandercammen | Species and tissue distribution of the regulatory protein of glucokinase[END_REF]. Surprisingly, several of the ligands of rat GKRP affected the etherase activity of xenopus GKRP (Fig. 4.C and 4.D), most particularly sorbitol 6-phosphate (Fig. 4.C), which acted with a similar affinity as on rat GKRP (I 50 = 30 M), but also mannose 6-phosphate (I 50 = 320 M) and fructose 6-phosphate (I 50 = 1 mM), which were poorer ligands for xenopus GKRP than for its rat counterpart. Remarkably, fructose 1phosphate, the best ligand of rat GKRP [START_REF] Detheux | Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation[END_REF], did not inhibit the etherase activity of xenopus GKRP (Fig. 4.D) whereas N-acetylglucosamine 6-phosphate inhibited it (I 50 = 130 M) more potently than that of the rat protein (Fig. 4.A and 4.C). Thus, unlike what was previously thought, xenopus GKRP shares some of the ligands of rat GKRP. Sorbitol 6-phosphate, ribose 5-phosphate and N-acetylglucosamine 6-phosphate modestly inhibited the activity of the N. gruberi GKRP homologue (Fig. 5). Except for Nacetylglucosamine 6-phosphate, none of the modulators of rat GKRP inhibited the activity of H. influenzae MurQ (Fig. 5), or of E. coli MurQ that was partially purified by DEAEchromatography from a bacterial cell extract (not shown). These findings indicated that the etherase activities of recombinant rat and xenopus GKRPs were authentically contributed by these proteins rather than by a contaminating E. coli etherase activity.

Effect of mutations of rat GKRP on its etherase activity

We have also tested the etherase activity of several rat GKRP mutants that had been produced and investigated in previous studies [START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF][START_REF] Veiga-Da-Cunha | Mutations in the glucokinase regulatory protein gene in 2p23 in obese French caucasians[END_REF]. The etherase activity was markedly reduced in three mutants (R518Q, K514A, T109A, S110A) in which residues that are highly conserved in MurQ/GKRP had been replaced (Table 2). Furthermore, the residual etherase activity of these hypoactive mutants (tested with 1.5 mM N-acetylmuramate 6-phosphate) was insensitive to fructose 6-phosphate, sorbitol 6-phosphate and fructose 1-phosphate (all tested at 5 mM; not shown), consistent with previous findings indicating that these mutants had a markedly reduced affinity for these phosphate esters in assays of GK inhibition. Reciprocally, the etherase activity was essentially unchanged in two other mutants (P446L, G107C) that had not lost their affinity for regulatory phosphate esters (Table 2; Fig. 6). These findings indicated that the catalytic site of the etherase activity and the site regulating the interaction with GK are one and the same.

Studies on the interaction of GKRPs and its homologues with GK

Since sorbitol 6-phosphate and mannose 6-phosphate are ligands of xenopus GKRP, we were interested to check whether they were also able to modulate the interaction between GK and xenopus GKRP. Fig. 7 compares the effect of these and other compounds on the inhibition exerted by rat or xenopus GKRPs on human islet or xenopus GK. In the case of rat GKRP, all assays were performed either in the absence (Fig. 7.A) or in the presence (Fig. 7.B) of 50 M fructose 6-phosphate. The results obtained with the rat protein are consistent with previously published data [START_REF] Detheux | Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation[END_REF], as they show that sorbitol 6-phosphate and fructose 6-phosphate strongly reinforced the inhibition, whereas mannose 6-phosphate, N-acetylmuramate 6-phosphate and N-acetylglucosamine 6-phosphate alleviated it (Fig. 7.B). The most remarkable observation is that none of these compounds affected the interaction between xenopus GKRP and GK (Fig.

7.C).

We also tested the effect of the N. gruberi GKRP homologue or H. influenzae MurQ on human and xenopus GK activity. No inhibition was found, even at concentrations of these proteins that were more than 10-fold higher than those at which rat or xenopus GKRP caused 50 % inhibition of GK (not shown).

DISCUSSION

Evolution of MurQ into GKRP

The findings presented in this paper indicate that MurQ underwent a gene duplication and fusion event, which first generated a new form of etherase, later to become GKRP in vertebrates. The main argument that we provide is that the GKRP/MurQ homologue of the primitive eukaryote N. gruberi, while having two MurQ domains and being closer to vertebrate GKRPs than to bacterial MurQ, is an efficient etherase, with a K cat that favourably compares with those of its bacterial homologues. A second argument is that both rat and xenopus GKRPs display some etherase activity, though this activity is more than two orders of magnitude lower than those of their bacterial or amoebal homologues.

This evolution is schematized in Fig. 8. A dimer comprising two symmetric catalytic sites has been replaced by a monomer with a single catalytic site and a hinge region. Steric hindrance in the hinge region is probably the reason why one of the two catalytic sites has been lost. Further evolution to "authentic" GKRPs involved mutations that led (1) to an improved binding of phosphate esters such as fructose 1-phosphate and fructose 6-phosphate, presumably at the expense of a lowering of the catalytic efficiency of the etherase reaction and (2) to the appearance of a binding site for GK.

The "catalytic site" of rat GKRP is indeed responsible for the binding of the allosteric ligands that favour or antagonize its inhibitory effect on GK. This is indicated by the finding that ligands able to modulate the interaction with GK also inhibit the etherase activity of rat GKRP. Moreover, their relative affinity in their effect on the etherase activity is comparable to that observed for the GK/GKRP interaction. Finally, mutations that affect the catalytic activity of rat GKRP also affect its capacity to bind regulatory phosphate esters.

The binding site of GKRPs for GK most probably comprises regions that are conserved between the rat and xenopus proteins, but not in bacterial MurQ or in the amoebal etherase. One such region is the N-terminal part, which is well conserved between xenopus and rat GKRPs (76 % identity for the first 25 residues, as compared to 59 % identity for the whole sequences), but is absent in the other proteins (Fig. 1). Accordingly, the presence of an N-terminal tag was shown to reduce the affinity of rat GKRP for GK [START_REF] Mookhtiar | Heterologous expression and characterization of rat liver glucokinase regulatory protein[END_REF], presumably because the tag sterically interferes with the binding of the N-terminal region of GKRP. Another region that has been proposed to play a role in the binding of GKRP to GK is the sequence LSApfVAG [START_REF] Baltrusch | Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner[END_REF]. This sequence, which is largely conserved in xenopus GKRP but less so in the N. gruberi GKRP homologue or in bacterial MurQ (see Fig. 1), is close to the Nterminal region in the 3D structure of MurQ [START_REF] Kim | Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling[END_REF]. We speculate that this sequence together with the N-terminal extension present in vertebrate GKRPs contribute to form the GK binding site. Other regions are probably also involved, since the GK/GKRP interaction is largely hydrophobic [START_REF] Anderka | Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors[END_REF]. Binding of phosphate esters presumably changes the relative position of these binding regions, thereby favouring or preventing GKRP interaction with GK.

The special case of xenopus GKRP

The effect of xenopus GKRP on the activity of GK has been extensively characterized [START_REF] Veiga-Da-Cunha | Cloning and expression of a Xenopus liver cDNA encoding a fructose-phosphate-insensitive regulatory protein of glucokinase[END_REF] and found to be insensitive to the classical effectors of rat and human GKRPs (fructose 6phosphate and fructose 1-phosphate), leading to the conclusion that xenopus GKRP was most likely unable to bind these effectors. The effects of sorbitol 6-phosphate, mannose 6phosphate, fructose 6-phosphate, and ribitol 5-phosphate on the etherase activity of xenopus GKRP all indicate that, on the contrary, this protein is able to bind these phosphate esters. The lack of response in terms of modulation of GK activity suggests that the interaction site with GK is uncoupled from the phosphate ester binding site, in xenopus GKRP and that this protein stays locked in an inhibitory conformation. We may, however, not exclude that ligands other than those we have tested are able to modulate this interaction.

Explanation for the low degree of identity between the two MurQ repeats of GKRP

The loss of the second catalytic site in eukaryotic MurQ homologues may explain the intriguing observation (schematized in Fig. 8) that the degree of identity between the two halves of rat GKRP (10.6% identity; 14) is significantly lower (p < 0.0001 and p = 0.0165 by chi square analysis) than for the comparisons between H. influenzae MurQ and either the Nterminal or the C-terminal half of rat GKRP (25.8 and 18.7 % identity, respectively). The low degree of identity between the two halves of GKRP has led Jaeger and Mayer [START_REF] Jaeger | The transcriptional factors MurR and catabolite activator protein regulate N-acetylmuramic acid catabolism in Escherichia coli[END_REF] to suggest that the duplication leading to the formation of eukaryotic GKRP is a very ancient event, predating the separation between GKRP and present day bacterial etherases. If this were true, only one of the two halves of GKRP (the one that separated last from the bacterial sequences) should show a higher degree of identity with bacterial MurQs, whereas the other half should show as little identity with bacterial MurQ as with the other half of GKRP. This is not the case and we therefore propose an alternative explanation. After the duplication/fusion event, the constraints imposed onto the two MurQ domains were different because only one of the two catalytic sites could be conserved. Thus, several residues that were not "allowed to mutate" in bacterial MurQ could mutate in the N-terminal part of the fusion protein, and a different set of residues could mutate in the C-terminal part, leading to the high degree of divergence between the two MurQ repeats in GKRP.

Physiological function of eukaryotic GKRP homologues

It is likely that all amoebal homologues of GKRP play physiologically the role of Nacetylmuramate 6-phosphate etherases. Amoeba prey on bacteria and it is therefore likely that they have enzymes allowing them to metabolize the muramylpeptide. Interestingly, their genomes encode homologues of anhydromuramate kinase, an enzyme serving to open the internal oside bond of 1,6-anhydro-N-acetylmuramate and phosphorylate the 6 th carbon of Nacetylmuramate [START_REF] Uehara | Recycling of the anhydro-N-acetylmuramic acid derived from cell wall murein involves a two-step conversion to N-acetylglucosamine-phosphate[END_REF].

At the other extreme of eukaryotic evolution, the rather high K m and low K cat values of the etherase activity of vertebrate GKRPs argues against a role of these proteins in the metabolism of N-acetylmuramate 6-phosphate. This is consistent with the fact that vertebrates do not have homologues of the enzyme that phosphorylates 1,6-anhydro-N-acetylmuramate. An enzyme that could potentially catalyse the phosphorylation of N-acetylmuramate to Nacetylmuramate 6-phosphate is N-acetylglucosamine kinase, most particularly since homologues of this enzyme form bifunctional proteins with MurQ homologues in fungi. However, a partially purified preparation rat liver N-acetylglucosamine kinase did not detectably phosphorylate N-acetylmuramate (Van Schaftingen, unpublished results). All this suggests that the capacity of vertebrates to metabolize N-acetylmuramate 6-phosphate is negligible. This is consistent with in vivo data obtained by injecting N-acetylmuramyl peptides to rats, which indicated rather poor and incomplete metabolism of these bacterial products [START_REF] Ono | Metabolic disposition of 14C-muroctasin in laboratory animals[END_REF].

As for the GKRP homologues present in invertebrates, it is difficult to decide about their function. None of these organisms possesses a 1,6-anhydro-N-acetylmuramate kinase, but, as suggested above, N-acetylglucosamine kinase could serve to phosphorylate Nacetylmuramate. Alternatively, the GKRP homologues could well have assumed another function, possibly that of controlling the activity of another enzyme than GK.

We do not see indeed any particular reason why GK is regulated by a protein that was originally a N-acetylmuramate 6-phosphate etherase, as there is no obvious link between Nacetylmuramate and GK. It may well be that GKRP plays other regulatory roles than that of controlling the activity and subcellular localisation of GK. Its involvement in the regulation of enzymes involved in lipid metabolism might, for instance, explain the linkage between SNPs in the GKRP gene and triglyceride levels.

FIGURE LEGENDS Figure 1. Alignment of GKRP and homologous bacterial and amoebal sequences

The GKRP sequences of X. laevis (GKRPXl) and R. norvegicus (GKRPRn) are aligned with the homologous sequences from N. gruberi (MurQNg) and H. influenzae (MurQHi1 and MurQHi2). The H. influenzae sequence is aligned twice, from residue 1 to 222 (MurQHi1) and from residues 25 to 303 (MuQHi2). The strictly conserved residues are shown in bold. The MurQ residues that are conserved in the N-terminal part of all three eukaryotic proteins are underlined twice in the second MurQ repeat. Those that are conserved in the C-terminal part of the eukaryotic proteins are underlined once in the first MurQ repeat. The LSApfVAG GK binding motif [START_REF] Baltrusch | Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner[END_REF] is underlined in rat GKRP.

Figure 2. Phylogenetic tree of GKRP

Phylogenetic tree was estimated from protein sequences using distance matrix neighbour joining (NJ) and Bayesian analysis (MrBaynes) methods. Posterior probabilities (MrBayes) or bootstrap support values (NJ) are indicated at the nodes. The horizontal bar represents 20 substitutions per 100 amino-acid residues.

Figure 3. Spatial localization in H. influenzae MurQ of the residues that are conserved in the N-and C-terminal halves of GKRP

The residues of the N-terminal half (A) and the C-terminal half (B) of rat GKRP that are conserved in H. influenzae MurQ are shown as blue or green spheres in the 3D structure of the MurQ monomer [START_REF] Kim | Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling[END_REF]. Those that are conserved in both regions are shown in yellow. The vertical line indicates the position of the rotational symmetry axis of the MurQ dimer. The two halves of GKRP have most likely the same spatial disposition as the bacterial dimer. The orange part in (A) corresponds to the missing amino acid residues at the end of the N-terminal half of GKRP. This region is fused to the C-terminal half to form a hinge. The position of the residues whose mutations are studied in the present work is also shown.

Figure 4. Inhibition of the etherase activity of rat GKRP (A and B) and xenopus GKRP (C and D) by various phosphate esters

The etherase activity of GKRPs was assayed by measuring the formation of D-lactate. Recombinant GKRPs (150 g/ml) were incubated at 37°C for 40 (rat GKRP; rGKRP) or 20 min (xenopus GKRP; xGKRP) in presence of subsaturating (350 M) N-acetylmuramate 6phosphate (MurNAc6P) and of the indicated concentrations of glucose 6-phosphate (Glc6P;

), fructose 6-phosphate (Fr6P; ), sorbitol 6-phosphate (Sol6P; ), ribose 5-phosphate (R5P; ), mannose 6-phosphate (Man6P; ), ribitol 5-phosphate (Rol5P; ), fructose 1phosphate (Fr1P; ) or N-acetylglucosamine 6-phosphate (GlcNAc6P; ). Values are means ± SEM (n = 3) and are expressed as percentage of the activity measured in the absence of effectors. The etherase activity of the indicated rat GKRPs (rGKRP) was tested by measuring the formation of D-lactate in the presence of 350 M N-acetylmuramate 6-phosphate (MurNAc6P; 100 % of initial activity) with or without 1 mM fructose 6-phosphate (Fr6P), fructose 1-phosphate (Fr1P) or sorbitol 6-phosphate (Sol6P). Values are means ± SEM (n = 3). (A) Human islet GK (hiGK) was assayed in the presence of 5 mM glucose, without or with 5 g/ml rat GKRP (rGKRP) and with or without 1 mM of the indicated phosphate esters. (B) Same as (A) except that 50 M fructose 6-phosphate was also present. (C) Xenopus GK (xGK) was assayed in the presence of 3 mM glucose, with or without 0.8 g/ml xenopus GKRP (xGKRP) and with or without 1 mM of the indicated phosphate esters. The values shown are the ratios (expressed in percent) of the activities observed with and without GKRP and are means ± SEM for n = 3.

Figure 8. Evolution of bacterial MurQ into eukaryotic GKRPs.

In the MurQ dimer, each catalytic site is formed by the two subunits with residues symbolized in black and white. A gene duplication event followed by fusion led to the N-terminal half losing some of the residues involved in the formation of one of the two catalytic sites. Further evolution led to a complete mutational loss of residues forming this site, while the other one was conserved, and became a regulatory site in mammalian GKRP. The asymmetrical mutation of residues in the two halves of GKRP results in their very low degree of identity because of divergent evolution, but maintains a higher degree of identity with bacterial MurQ. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 1 Kinetic characteristics of the etherase activity of H. influenzae MurQ, N. gruberi GKRP homologue and xenopus and rat GKRPs

The etherase activity of H. influenzae MurQ (MurQHi) or N. gruberi GKRP homologue (MurQNg) (0.25 g/ml) was assayed with variable concentrations (10 to 200 M) of Nacetylmuramate 6-phosphate (MurNAc6P) by monitoring the formation of Nacetylglucosamine 6-phosphate. The etherase activities of xenopus (xGKRP) and rat (rGKRP) GKRPs (150 g/ml) were measured by monitoring the formation of D-lactate in the presence of 50 M to 1 mM N-acetylmuramate 6-phosphate. Assays were performed in triplicate and values shown are the means ± standard error.

K m (MurNAc6P) ( M) K cat (sec -1 ) K cat /K m (sec -1 . M -1 )
V max ( mol.min -1 .mg A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 12 Aug 2009 as manuscript BJ20090986 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090986A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  polymerase. The amplified DNA (1855 bp) without the stop codon was digested with NcoI and NotI and directly cloned in pET-28a. The open reading frames of the genes coding for Silicibacter pomeroyi N-acetylglucosamine kinase (YP_167078.1; 890 bp), glucosamine 6phosphate deaminase/isomerase (YP_167080; 1032 bp) and N-acetylglucosamine 6phosphate deacetylase (YP_167081; 1146 bp) were PCR-amplified using bacterial genomic DNA and Pfu polymerase. The PCR-amplified products containing the ATG codon in a NdeI restriction site and the stop codon flanked by a BamHI (N-acetylglucosamine kinase and glucosamine 6-phosphate deaminase/isomerase) or a BglII (N-acetylglucosamine 6phosphate deacetylase) restriction site were cloned in pET15b. The sequence of the primers used in the PCR reactions are shown in supplementary Table

Biochemical

  Journal Immediate Publication. Published on 12 Aug 2009 as manuscript BJ20090986 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090986

Biochemical

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  Biochemical Journal Immediate Publication. Published on 12 Aug 2009 as manuscript BJ20090986 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090986A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Biochemical

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Figure 5 .

 5 Figure 5. Inhibition of the etherase activity of H. influenzae MurQ and N. gruberi GKRP homologue by various phosphate esters

Figure 6 .

 6 Figure 6. Inhibition of the etherase activity of wild type and active mutant forms of rat GKRP by various phosphate esters.

Figure 7 .

 7 Figure 7. Effect of sorbitol 6-phosphate, mannose 6-phosphate, N-acetylglucosamine 6phosphate and N-acetylmuramate 6-phosphate on the inhibition exerted by rat and xenopus GKRPs on GK .

Biochemical

  Journal Immediate Publication. Published on 12 Aug 2009 as manuscript BJ20090986 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090986

Figure 1 (Figure 2 (Figure 3 .Figure 3 . 24 BiochemicalFigure 4 (

 1233244 Figure 1 (Veiga-da-Cunha et al.)

Figure 4 .Figure 5 (Figure 5 .Figure 6 (Figure 6 .Figure 7 (Figure 7 .Figure 8 (

 45566778 Figure 4.pzf:Fig.4 -Mon Jun 29 11:20:00 2009

Table 2 Effect of mutations of the catalytic and regulatory properties of rat GKRP The

 2 etherase activities were measured by monitoring the formation of D-lactate in the presence of 350 M to 1.4 mM N-acetylmuramate 6-phosphate (MurNAc6P). Assays were performed in triplicate and values shown are the means ± standard error. n.d.; not determined.

	Rat GKRP	K m (MurNAc6P) (mM)	V max (MurNAc6P) (nmol.min -1 .mg -1 )	K d (F6P) ( M)	K d (Sol6P) ( M)	K d (Fr1P) ( M)
	Wild-type a	0.70 ± 0.11	44 ± 3	20	5	1
	P446L b	0.56 ± 0.20	38 ± 6	20	5	1
	G107C a	0.73 ± 0.07	48 ± 2	1	0.4	29
	R518Q b	1.36 ± 0.64	20 ± 5	830	80	1600
	K514A a	2.24 ± 0.36	13 ± 1	>2000	n.d.	n.d.
	S110A a	1 ± 0.40	11 ± 2	>2000	300	100
	T109A a	n.d.	1.33 ± 0.17 c	40	2.9	3.6
					inversion of
					effect

a The values of Kd(Fr6P), Kd(Sol6P), Kd(Fr1P) are from

[START_REF] Veiga-Da-Cunha | Identification of fructose 6phosphate-and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase[END_REF]

b The values of Kd(Fr6P), Kd(Sol6P), Kd(Fr1P) are from

[START_REF] Veiga-Da-Cunha | Mutations in the glucokinase regulatory protein gene in 2p23 in obese French caucasians[END_REF]

c Etherase activity at 1.

[START_REF] Brocklehurst | Differences in regulatory properties between human and rat glucokinase regulatory protein[END_REF]

mM MurNAc6P Biochemical Journal Immediate Publication. Published on 12 Aug 2009 as manuscript BJ20090986 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090986

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACNOWLEDGEMENTS

We thank J. De Jonckheere for the kind gift of N. gruberi DRI genomic DNA and Kate Peel for her expert technical help.

FUNDING

This work was supported by grants from the Belgian Fonds National de la Recherche Scientifique and the Interuniversity Attraction Poles Programme -Belgian Science Policy. Maria Veiga-da-Cunha is Chercheur Qualifié of the Belgian Fonds National de la Recherche Scientifique. Biochemical Journal Immediate Publication. Published on 12 Aug 2009 as manuscript BJ20090986 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090986

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Izabela Koszela-Piotrowska # # # #

Karolina Matkovic # # # #

Adam Szewczyk

Wieslawa Jarmuszkiewicz

A Large-Conductance Calcium-Activated Potassium Channel in Potato Tuber Mitochondria

Keywords: , bovine serum albumin, IbTx, iberiotoxin, KCOs, potassium channel openers, mitoBK Ca channel, mitochondrial large-conductance Ca 2+ -activated potassium channel, mitoK ATP channel, mitochondrial ATP-sensitive potassium channel, NS1619, SMP, submitochondrial particles, TPP + , tetraphenylphosphonium ∆Ψ, mitochondrial transmembrane bioenergetics, electrophysiology, mitochondria, potassium channel, Solanum tuberosum

In the present study, we describe the existence of a novel potassium channel in the plant (potato tuber) mitochondrial inner membrane. We found that substances known to modulate large-conductance calciumactivated potassium channel activity influenced the bioenergetics of potato tuber mitochondria. In isolated mitochondria, Ca 2+ and NS1619 (a potassium channel opener) were found to depolarize the mitochondrial membrane potential and to stimulate resting respiration. These effects were blocked by iberiotoxin (a potassium channel inhibitor) in a potassium-dependent manner. Additionally, the electrophysiological properties of the large-conductance potassium channel present in the potato tuber inner mitochondrial membrane are described in a reconstituted system, using planar lipid bilayers. After incorporation in 50/450 mM KCl gradient solutions, we recorded large-conductance potassium channel activity with conductance from 502 ± 15 to 615 ± 12 pS. The probability of channel opening was increased by Ca 2+ and reduced by iberiotoxin. Immunological analysis with antibodies raised against the mammalian plasma membrane largeconductance Ca 2+ -dependent K + channel identified a pore-forming α subunit and an auxiliary β2 subunit of the channel in potato tuber mitochondrial inner membrane. These results suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.

INTRODUCTION

Mitochondria play an important role in energy metabolism within the cell. In addition to this canonical function, mitochondria are involved in intracellular signaling. Recently, potassium transport through the mitochondrial inner membrane was found to play a central role in the cytoprotection of various mammalian cell types [START_REF] O'rourke | Evidence for mitochondrial K+ channels and their role in cardioprotection[END_REF][START_REF] Szewczyk | Mitochondrial potassium channels[END_REF]. This transport is strictly ion channel-dependent. Similarly to the plasma membrane, potassium-selective ion channels are found in the inner mitochondrial membrane [START_REF] Szewczyk | The intracellular potassium and chloride channels: properties, pharmacology and function[END_REF][START_REF] Bernardi | Mitochondrial transport of cations: channels, exchangers, and permeability transition[END_REF][START_REF] Szewczyk | Mitochondria as a pharmacological target[END_REF]. Potassium ions control mitochondrial metabolism, primarily via a regulation of matrix volume [START_REF] Halestrap | Regulation of mitochondrial metabolism through changes in matrix volume[END_REF]. The biophysical and pharmacological properties of mitochondrial potassium channels are similar to some of the potassium channels present in the plasma membrane of various mammalian cell types, for example mitochondrial ATP-regulated potassium channels (mitoK ATP channels) [START_REF] Inoue | ATP-sensitive K + channel in the mitochondrial inner membrane[END_REF], large-conductance calcium-activated potassium channels (mitoBK Ca channels) [START_REF] Siemen | Ca 2+ -activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line[END_REF], and voltage-dependent potassium Kv1.3 channels [START_REF] Szabo | A novel potassium channel in lymphocyte mitochondria[END_REF].

In plant mitochondria, the existence of different energy-dissipating systems is necessary for metabolic regulation [START_REF] Jarmuszkiewicz | Alternative oxidase and uncoupling protein: thermogenesis versus cell energy balance[END_REF]. Plant mitochondria (but not animal mitochondria) contain an alternative oxidase (AOX) that catalyses ubiquinol-oxygen oxidoreduction without H + release into the cytosol, thus dissipating the redox potential energy [START_REF] Sluse | Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role[END_REF][START_REF] Siedow | The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity[END_REF]. Similarly to animal mitochondria, plant mitochondria contain an uncoupling protein (PUMP) that dissipates a proton electrochemical gradient by mediating re-entry of H + into the mitochondrial matrix in the presence of free fatty acids [START_REF] Vercesi | Plant uncoupling mitochondrial proteins[END_REF]. Another energy-dissipating system present in plant mitochondria (as in animal mitochondria) is the ATP-sensitive potassium channel (mitoK ATP channel), which mediates an electrophoretic K + uniport, likely working together with an electroneutral K + /H + exchanger [START_REF] Pastore | The existence of the K + channel in plant mitochondria[END_REF][START_REF] Petrussa | Cyclosporin A induces the opening of a potassium-selective channel in higher plant mitochondria[END_REF][START_REF] Chiandussi | Modulation of a plant mitochondrial K + ATP channel and its involvement in cytochrome c release[END_REF][START_REF] Diolez | Correlations between ATP-synthesis, membrane potential and oxidation rate in plant mitochondria[END_REF]. Descriptions of the plant mitoK ATP channel have been based mainly on studies of isolated mitochondria in which the influence of K + entry into the mitochondrial matrix on membrane potential (∆Ψ) dissipation and mitochondrial swelling were assessed. The plant mitoK ATP channel seems to be sensitive to the same potassium channel modulators as the mammalian mitoK ATP channel. In durum wheat and pea mitochondria, the mitoK ATP channel is activated by diazoxide and GTP, and its activity is inhibited by glyburide and 5-hydroxydecanoate (in pea mitochondria) [START_REF] Pastore | The existence of the K + channel in plant mitochondria[END_REF][START_REF] Chiandussi | Modulation of a plant mitochondrial K + ATP channel and its involvement in cytochrome c release[END_REF][START_REF] Pastore | Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria[END_REF]. Moreover, the plant mitoK ATP appears to be stimulated by cyclosporin A and regulated by dithiol-disulfide interconversion, H 2 O 2 , and NO [START_REF] Petrussa | Cyclosporin A induces the opening of a potassium-selective channel in higher plant mitochondria[END_REF][START_REF] Chiandussi | Modulation of a plant mitochondrial K + ATP channel and its involvement in cytochrome c release[END_REF][START_REF] Casolo | Involvement of the mitochondrial K + ATP channel in H 2 O 2 -or NO-induced programmed death of soybean suspension cell cultures[END_REF]. On the other hand, an ATP-insensitive, quinine-inhibited potassium import pathway has been described in potato, tomato, and maize mitochondria [START_REF] Ruy | A Highly active ATP-insensitive K+ import pathway in plant mitochondria[END_REF].

So far, there has been no information about other possible plant mitochondrial potassium channels, such as the large-conductance Ca 2+ -activated potassium channel (mitoBK Ca channel) described in animal mitochondria. Therefore, the aim of this study was to search for a mitoBK Ca channel in plant mitochondria and to determine the bioenergetic consequences of its activation. For this purpose, we studied the effects of BK Ca channel activators (Ca 2+ , NS1619) and inhibitor (iberiotoxin, IbTx) on ∆Ψ and respiration in isolated potato tuber mitochondria. Moreover, measurements of the electrophysiological properties of a single mitoBK Ca channel in a reconstituted system were carried out and immunological detection experiments were performed to confirm the discovery of a plant mitoBK Ca channel.

EXPERIMENTAL Chemicals

L-α-phosphatidyl-choline (asolectin), NS1619, and n-decane were purchased from Sigma-Aldrich, and IbTx from Bachem. All other chemicals were of the highest purity commercially available. NS1619 was dissolved in methanol, and IbTx in H 2 O. again at 1000 x g for 5 min and then at 11000 x g for 20 min. Mitochondria were subsequently purified on a self-generating 22% Percoll gradient (at 40000 x g for 30 min). Purified mitochondria were washed twice in the same medium without EGTA and BSA. The mitochondrial protein concentration was determined by the biuret method.

Preparation of membrane fractions

To obtain submitochondrial particles (SMP), mitochondria were isolated as above, but with media of K + salts. Freshly prepared mitochondria were diluted to about 8 mg of protein/ml with 0.3 M sucrose and 10 mM Hepes-KOH, pH 7.2, and subsequently frozen at -20ºC. After thawing, the suspension was sonicated 6 times for 30 s and centrifuged at 12000 × g for 15 min to pellet the unbroken mitochondria. The supernatant was centrifuged at 200000 × g (2 h) to pellet SMP. Fractions of SMP, which are the mitochondrial inner membrane-enriched fractions, were resuspended in 0.3 M sucrose and 10 mM Hepes-KOH, pH 7.2 at a concentration of 5 mg of protein/ml.

To obtain a plasma membrane-enriched fraction, the supernatant from the first 11000 x g centrifugation during mitochondria isolation was centrifuged at 100000 x g for 1h. The pellet was resuspended in 0.3 M sucrose and 10 mM Hepes-KOH, pH 7.2.

Planar lipid membrane (PLM) measurements

Experiments were performed with potato tuber SMP, as described previously [START_REF] Bednarczyk | Quinine inhibits mitochondrial ATP-regulated potassium channel from bovine heart[END_REF][START_REF] Hordejuk | pH modulation of large conductance potassium channel from adrenal chromaffin granules[END_REF][START_REF] Bednarczyk | Matrix Mg 2+ regulates mitochondrial ATPdependent potassium channel from heart[END_REF]. In brief, PLMs were formed in a 250-µm-diameter hole drilled in a Delrin cup (Warner Instrument, CT), which separates the two chambers (cis and trans, each with a 1-ml internal volume). The chambers contained 50/450 mM KCl (trans/cis) and 20 mM Tris-HCl solutions, pH 7.2. The outline of the aperture was coated with a lipid solution and N 2 dried prior to bilayer formation to improve membrane stability. PLMs were painted using asolectin in n-decane at a final concentration of 25 mg of lipid/ml. SMP (about 3 µg of protein/ml, 0.5-1.5 µl/reconstitution) were added to the trans compartment (Figure 5A). All measurements were carried out at room temperature (~25ºC). Formation and thinning of the bilayer were monitored by capacitance measurements and optical observations. Final accepted capacitance values ranged from 120 to 230 picofarads. Electrical connections were created using Ag/AgCl electrodes and agar salt bridges (3 M KCl) to minimize liquid junction potentials. Voltage was applied to the cis compartment of the chamber, and the trans compartment was grounded. The current was measured using a bilayer membrane amplifier (BLM-120, BioLogic). Signals were low-pass filtered at 500 Hz. The current was digitized at a sampling rate of 100 kHz (A/D converter PowerLab 2/20, ADInstruments) and transferred to a PC for off-line analysis by Chart version 5.5.5 (PowerLab ADInstruments) and pCLAMP10.2 (Axon Instruments). The pCLAMP10.2 software package was used for data processing. The channel recordings presented are representative of the most frequently observed conductance values under the given conditions. Ion conductance was calculated from the linear regression of the points in the current-voltage relationship using the GraphPad Prism 4 program.

Mitochondrial oxygen consumption

Oxygen uptake was measured polarographically with a Clarck type oxygen electrode (Rank Brothers) in 3 ml of standard incubation medium (25°C) consisting of 0.35 M mannitol, 3 mM NaH 2 PO 4 , 1 mM MgCl 2 , 10 mM TrisHCl, 10 mM MES, pH 6.8, and 0.1% BSA. Changes in the composition of the incubation medium are described in the figure legend. Measurements were performed with 0.8-1 mg of mitochondrial protein in the presence of 1.5 mM benzohydroxymate (an inhibitor of alternative oxidase), 0.14 mM ATP (to activate succinate dehydrogenase), 1.8 µM carboxyatractylozide (to exclude ATP/ADP antiporter activity), and 10 mM glibenclamide (to inhibit the mitoK ATP channel). Succinate (5 mM) plus rotenone (2 µM) was used as a respiratory substrate. Respiratory rate measurements were performed in the absence of added ADP, i.e., in the resting state (state 4). State 3 (phosphorylating) respiration measurements were performed to check the coupling parameters. Only high quality mitochondria preparations, i.e., with an ADP/O value of around 1.40 and a respiratory control ratio of around 2.5-3 (with succinate), were used in all experiments. O 2 uptake values are presented in nmol O × min -1 × mg -1 protein. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Mitochondrial membrane potential measurements

The mitochondrial ∆Ψ was measured simultaneously with oxygen uptake using a tetraphenylphosphonium (TPP + )-specific electrode according to Kamo et al. [START_REF] Kamo | Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state[END_REF]. Measurements were performed in the presence of 6 µM TPP + . To calculate the ∆Ψ value, the matrix volume of potato tuber mitochondria was assumed to be 2.0 µl × mg -1 protein. The calculation assumes that the TPP + distribution between mitochondria and medium followed the Nernst equation. Corrections were made for TPP + binding to mitochondrial membranes. Values of ∆Ψ are presented in mV.

SDS-PAGE and immunoblotting

Samples of isolated mitochondrial proteins, SMP or the plasma membrane-enriched fraction were solubilized in sample buffer containing 2% (w/v) SDS, 50 mM Tris/Cl (pH.6.8), 10% glycerol, 0.004% (w/v) bromophenol blue, and 8% mercaptoethanol, and then boiled for 4 min. Proteins were separated in 12.5% SDS-polyacrylamide gels, and then electrotransferred to a nitrocellulose membrane. Membranes were then hybridized with anti-K Ca 1.1 and anti-sloβ2 antibodies (APC-107 and APC-034, Alomone) at dilutions of 1:500 or 1:200, respectively, in the presence or absence of blocking peptide. Cross reactivity was also checked with antibodies raised against a plant plasma membrane H + -ATPase (at a dilution of 1:1000). Protein detection was achieved with secondary antibodies linked to horseradish peroxidase (at a dilution of 1:20000) and the ECL chemiluminescence system. Protein content was determined by the Bradford method (Bio-Rad).

RESULTS

Effect of potassium channel modulators on respiratory rate and membrane potential in isolated potato tuber mitochondria

To investigate the possible effects of Ca 2+ on the potassium permeability of isolated potato tuber mitochondria, we measured mitochondrial resting (nonphosphorylating) respiration and ∆Ψ in potassiumcontaining medium in the presence of different Ca 2+ concentrations, using succinate as an oxidizable substrate (Figure 1). An example of simultaneous measurements of the respiratory rate and ∆Ψ is shown in Figure 1A. Addition of Ca 2+ up to a concentration of 1 mM caused an increased respiration rate of up to 20 ± 2% in the absence of 1.5 µM IbTx, and up to 5 ± 3% in its presence (n = 4, S.D.) (Figure 1C). At the same time, ∆Ψ decreased after addition of Ca 2+ by up to 6.8 ± 0.5 mV and 0.7 ± 0.4 mV in the absence and presence of IbTx, respectively (n = 4, S.D.) (Figure 1D). Thus, the inhibitor previously described as specific for the mammalian large-conductance potassium channel [START_REF] O'rourke | Evidence for mitochondrial K+ channels and their role in cardioprotection[END_REF][START_REF] Szewczyk | Mitochondrial potassium channels[END_REF] considerably blocks Ca 2+ -induced respiration and ∆Ψ depolarization in isolated potato tuber mitochondria. These results suggest that Ca 2+ stimulates IbTx-sensitive K + flux into potato tuber mitochondria, decreasing ∆Ψ and thus accelerating the mitochondrial respiration rate. This indicates that the mitoBK Ca channel is present in potato tuber mitochondria. The Ca 2+ -induced IbTx-sensitive respiratory rate (i.e., the difference between the respiratory rate in the presence of Ca 2+ and the rate in the presence of Ca 2+ plus inhibitor) should represent mitoBK Ca channel activity (i.e., the channel-mediated K + flux). This activity, as measured using various Ca 2+ concentrations (Figure 1B), revealed that 50% of the maximal stimulation (S 0.5 ) by Ca 2+ is reached at 0.45 mM, as calculated from the linear regression of a double reciprocal plot (Figure 1B, inset). A similar value was attained when the Ca 2+ -induced IbTx-sensitive depolarization of ∆Ψ versus the Ca 2+ concentration was analyzed (data not shown). The partial insensitivity of Ca 2+ -induced respiration and ∆Ψ depolarization to IbTx observed in potato tuber mitochondria is likely related to electrophoretic Ca 2+ influx pathways present in some plant mitochondria [START_REF] Silva | Calcium transport by corn mitochondria: evaluation of the role of phosphate[END_REF] or permeability transition opening [START_REF] De Oliveira | Cyclosporin A inhibits calcium uptake by Citrus sinensis mitochondria[END_REF].

The potassium channel opener NS1619, previously described to be specific for the mitoBK Ca channel [START_REF] O'rourke | Evidence for mitochondrial K+ channels and their role in cardioprotection[END_REF][START_REF] Szewczyk | Mitochondrial potassium channels[END_REF], was also used in our studies. Similarly to calcium ions, NS1619 stimulates mitochondrial resting oxygen uptake and decreases mitochondrial resting ∆Ψ (Figure 2). An example of the effect of increasing concentrations of NS1619 on respiratory rate and ∆Ψ is shown in Figure 2A. Addition of NS1619 up to 40 µM resulted in an increase in the rate of respiration up to 100 ± 6% and 53 ± 7% (n = 6, S.D.) in the A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

absence and presence of 1.5 µM IbTx, respectively (Figure 2C). At the same time, ∆Ψ decreased after addition of up to 40 µM NS1618 by up to 18.1 ± 2 mV and 10.6 ± 1.3 mV (n = 6, S.D.) in the absence and presence of IbTx, respectively (Figure 2D). Thus, IbTx partially blocks (approximately 50%) NS1619induced respiration and ∆Ψ depolarization in isolated potato tuber mitochondria. When compared to the effect of IbTx on Ca 2+ -induced respiration and ∆Ψ depolarization (Figure 3), this could indicate a nonspecific uncoupling effect of NS1619 on isolated potato tuber mitochondria rather than low sensitivity of the channel to IbTx. Similarly, the sensitivity of NS1619-induced respiration and ∆Ψ depolarization to the mitoBK Ca channel blockers (IbTx, charybdotoxin, paxilline) in some mammalian isolated mitochondria is not complete [START_REF] Heinen | Mitochondrial Ca 2+ -induced K + influx increases respiration and enhances ROS production while maintaining membrane potential[END_REF][START_REF] Heinen | Physiological levels of glutamine prevent morphine-induced preconditioning in the isolated rat heart[END_REF][START_REF] Skalska | A novel potassium channel in skeletal muscle mitochondria[END_REF][START_REF] Skalska | Calcium ions regulate K uptake into brain mitochondria: the evidence for a novel potassium channel[END_REF]. Nevertheless, we can conclude that in potato tuber mitochondria, NS1619 stimulates IbTx-sensitive K + flux, decreases ∆Ψ, and thus accelerates the mitochondrial respiration rate, further indicating the presence of the mitoBK Ca channel activity. The NS1619-induced IbTx-sensitive respiratory rate (i.e., the difference between the respiratory rate in the presence of the modulator and the rate after addition of the inhibitor) measured at various NS1619 concentrations revealed that 50% of maximal stimulation (S 0.5 ) is reached at 25 µM NS1619 [Figure 2B, inset]. When NS1619-induced IbTx-sensitive depolarization of ∆Ψ at various NS1619 concentrations was plotted, a similar value of S 0.5 for NS1619 was obtained (data not shown).

Figure 3 illustrates the efficiency of IbTx in the inhibition of mitoBK Ca channel activity under unstimulated (no activators) or stimulated (in the presence of 0.8 mM CaCl 2 or 40 µM NS1619) conditions. An example of the effect of increasing concentrations of IbTx on NS1619-induced respiration and ∆Ψ depolarization is shown in Figure 3A. Under all studied conditions, addition of IbTx up to a concentration of 1.5 µM blocked unstimulated, NS1619-induced, and Ca 2+ -induced respiration by up to 4%, 53%, and 79%, respectively (Figure 3C). Similarly, IbTx restored up to 1%, 46% and 78% of unstimulated, NS1619induced and Ca 2+ -induced ∆Ψ depolarization, respectively (Figure 3D). These results indicate low activity of the mitoBK Ca channel in the absence of modulators in isolated potato tuber mitochondria, as IbTx has a slight inhibitory effect. Moreover, the results reveal that IbTx inhibits Ca 2+ -induced mitoBK Ca channel activity (~80% inhibition) more efficiently than NS1619-induced channel activity (~50% inhibition) again indicating a non-specific uncoupling effect of NS1619 on isolated potato tuber mitochondria. The relationship between NS1619-induced IbTx-sensitive respiration and various IbTx concentrations reveals that 50% of maximal inhibition (I 0.5 ) by IbTx is reached at 615 nM, as calculated from the linear regression of a double reciprocal plot (Figure 3B, inset). A similar value was obtained when the effect of increasing IbTx concentration on ∆Ψ depolarization was analyzed (data not shown).

To exclude an influence of the applied modulators (Ca 2+ , Ns1619, IbTx) on succinate dehydrogenase activity, experiments were also performed with external NADH as a respiratory substrate (data not shown). The results confirm observations taken from the experiments described above (Figures 123).

The findings presented herein strongly suggest the presence of the Ca 2+ -activated potassium channel in the inner membrane of potato tuber mitochondria. To further test this hypothesis, we investigated the effect of Ca 2+ and IbTx on isolated potato tuber mitochondria respiring (with succinate) in incubation media containing different monovalent cations (chloride salts) compared to those incubated in the presence of K + (Figure 4) or in the absence of any monovalent cations. In medium deprived of monovalent cations, no Ca 2+ -induced IbTx-sensitive increase in respiration and ∆Ψ depolarization were observed (data not shown). Figure 4 shows the influence of increasing concentrations (up to 50 mM) of KCl, NaCl, LiCl, RbCl, and CsCl on Ca 2+ -induced IbTx-sensitive ∆Ψ depolarization (i.e., the difference between ∆Ψ in the presence of 0.8 mM CaCl 2 and ∆Ψ in the presence of CaCl 2 and 1.5 µM IbTx). The results indicate that the influence of potassium channel modulators (Ca 2+ and IbTx) on isolated potato tuber mitochondria in the above experiments can be significantly attributed to K + influx through the inner membrane.

Electrophysiological properties of mitoBK Ca channel from the potato tuber mitochondrial membrane

The inner mitochondrial membrane-enriched SMP from potato tuber mitochondria were reconstituted into PLMs. Incorporation of channels into the bilayer was usually observed within 10-20 min after SMP addition to the trans compartment. SMP fractions from five different preparations were used. More than Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. 140 reconstitutions were monitored in the single-channel experiments. Approximately 40% of the channels incorporated into bilayers showed properties of the large-conductance potassium channels. Figure 5 shows representative current-time traces (Figure 5B) and current-voltage relationship (Figure 5C) for potassium channel opening at different voltages in the 50/450 mM KCl (cis/trans) gradient solutions. The current was measured as a function of applied potential at intervals ranging from +90 mV to -30 mV (Figure 5C). The calculated ion conductance is equal to 502 ± 15 pS for potential from -30 to 20 mV and 615 ± 12 pS for potential from +40 to 90 mV, thus indicating large-conductance potassium channel activity. The reversal potential of +34 mV calculated from curve fitting to the experimental data indicates that the examined ion channel is cation-selective. All incorporated large-conductance potassium channels were more active at positive than at 0 mV potentials (Figure 5D). Sometimes we recorded spontaneous closing of channels at 0 mV and at negative potentials (Figure 5B). The activity of such channels could be rescued after applying positive potentials, for example 50 mV.

Substances known to modulate mammalian mitoBK Ca channel activity were used to examine the properties of the K + ion channel observed in our experiments. Figure 6 demonstrates the effect of 300 µM Ca 2+ on the potato tuber mitochondrial large-conductance potassium channels. Figure 6A shows single channel recordings in the 50/450 mM KCl (cis/trans) gradient solutions at 0 mV, before and after addition of Ca 2+ . Ca 2+ caused a transition into an open state that indicates the existence of the mitoBK Ca channel in the potato tuber inner mitochondrial membrane. As depicted in the histograms shown in Figure 6B, the number of observations of the channel being in an open state changed from 73% to 86%. The probability of channel opening (P) at 0 mV potential increased from 0.49 ± 0.07 to 0.84 ± 0.05 (n = 4, p<0.0076, unpaired t test) in the presence of Ca 2+ (Figure 6C).

Figure 7 presents the inhibitory effect of IbTx on the potato tuber mitochondrial large-conductance potassium channels. As shown in the representative single channel recording obtained in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV, before and after the addition of 600 nM IbTx, the inhibitor caused a complete transition of the channel protein into a closed state (Figure 7A). The number of open state events changed from 83% to zero after IbTx addition, as illustrated in the histograms (Figure 7B). The probability of channel opening (P) at 0 mV potential decreased from 0.75 ± 0.27 for control recordings to 0.049 ± 0.04 after addition of 600 nM IbTx (p<0.008, unpaired t test). Moreover, IbTx blocked the channel activity in a dose-dependent manner (in a range from 200 to 600 nM) (Figure 7C). The apparent concentration of IbTx that provided half maximal inhibition (I 0.5 ) was approximately 170 nM. In our PLM experiments, 25% of the channels with a large conductance did not display sensitivity to IbTx (data not shown).

Immunological detection of plant mitoBK Ca channel proteins

Immunoblotting of total mitochondrial proteins, as well as SMP, allowed immunological detection of the plant mitoBK Ca channel. For this, antibodies raised against the mammalian plasma membrane BK Ca channel pore (α subunit K Ca 1.1) were used. In plant mitochondrial and SMP fractions, a protein band with a molecular mass of approximately 70 kDa was detected using the anti-K Ca 1.1 antibodies (Figure 8A). We also used antibodies raised against the mammalian plasma membrane BK Ca channel β subunits. In potato tuber mitochondrial and SMP fractions, anti-sloβ2 antibodies cross-reacted with a single band at around 30 kDa (Figure 8B). Less sensitive reactivity was observed with anti-sloβ1 and anti-sloβ4 antibodies (data not shown). As shown in Figure 8, much stronger signals were obtained with SMP compared to isolated mitochondria using anti-K Ca 1.1 and anti-sloβ2 antibodies, proving that the detected proteins localized to the inner membrane of potato tuber mitochondria. Moreover, specific blocking peptides blocked the antibodyantigen interaction, demonstrating the specificity of the reaction in the western blot analysis. On the other hand, mitochondrial (isolated mitochondria and SMP) and plasma membranes probed with antibodies to a plant plasma membrane marker (H + -ATPase) displayed no signal in the mitochondrial fractions, indicating the absence of surface membrane contamination (Figure 8C). Therefore, we can conclude that the potato tuber mitoBK Ca channel may contain subunits similar to the α subunit K Ca 1.1 and the β subunit sloβ2.

DISCUSSION

In the present study, we describe for the first time (to our knowledge) the functional properties of the mitoBK Ca channel-like protein in plant mitochondria. Potassium channel activators, Ca 2+ and NS1619, are able to modulate the resting respiratory rate (stimulation) and ∆Ψ (depolarization) in isolated potato tuber mitochondria. The opposite responses (respiratory rate inhibition and ∆Ψ repolarization) were observed when the mitoBK Ca channel inhibitor, IbTx, was applied. These effects were dependent on the presence of K + in the incubation medium. Thus, we provide evidence that the observed effects of the mitoBK Ca channel openers are due to activation of electrogenic potassium transport through the inner mitochondrial membrane, likely mediated by a potassium channel belonging to the family of mitochondrial potassium channels previously described in mammalian mitochondria [START_REF] Inoue | ATP-sensitive K + channel in the mitochondrial inner membrane[END_REF][START_REF] Siedow | The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity[END_REF]. This is the first study to investigate the electrophysiological properties of the large-conductance potassium channels in plant mitochondria. We used the planar lipid bilayers technique to confirm that large potassium channels are present in the potato tuber inner mitochondrial membrane. The cation selectivity, large conductance (502-615 pS), reverse potential (+34 mV) and sensitivity to Ca 2+ and IbTx of the potato tuber mitoBK Ca channel indicate similarity to the mammalian mitoBK Ca channels previously reported in glioma [START_REF] Siemen | Ca 2+ -activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line[END_REF], skeletal muscle [START_REF] Skalska | A novel potassium channel in skeletal muscle mitochondria[END_REF], brain [START_REF] Skalska | Calcium ions regulate K uptake into brain mitochondria: the evidence for a novel potassium channel[END_REF], and cardiac [START_REF] Xu | Cytoprotective role of Ca 2+ -activated K + channels in the cardiac inner mitochondrial membrane[END_REF] mitochondria. However, the ion conductance of the potato tuber mitoBK Ca channel (502-615 pS) is higher than that observed for mammalian mitoBK Ca channels (~260-300 pS). This could be due to highly active K + transport in some plant mitochondria, leading to enormous mitochondrial depolarization (also in potato tuber mitochondria) [START_REF] Pastore | The existence of the K + channel in plant mitochondria[END_REF], which contrasts with mammalian mitochondria in which ∆Ψ dissipation seems to be weaker [START_REF] Debska | Opening of potassium channels modulates mitochondrial function in rat skeletal muscle[END_REF][START_REF] Debska | Potassium channel openers depolarize hippocampal mitochondria[END_REF]. Moreover, mammalian plasma membrane BK Ca channel has a higher relative permeability for Rb + than for Na + [START_REF] Blatz | Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle[END_REF] in contrast to that observed for potato tuber mitoBK Ca channel. However, K + flux is dominant.

In addition to the functional studies, the presence of mitoBK Ca channel proteins in potato tuber mitochondria is indicated by their cross-reactivity with antibodies raised against the K Ca 1.1 α subunit of the mouse BK Ca channel and the β subunit of the human sloβ2 BK Ca channel. This suggests that the mitoBK Ca channel present in the potato tuber inner mitochondrial membrane may be structurally similar to the mammalian BK Ca channel. Namely, it may be formed by the principle pore-forming α subunit that interacts with an auxiliary β2 subunit. The predominant β2 subunit may determine the channel's activity, including its sensitivity to Ca 2+ and other modulators. The relative molecular mass of the detected potato tuber proteins (around 70 and 30 kDa for the α and β2 subunits, respectively) differs from that of the mammalian proteins from plasma membrane (~125 and ~44 kDa for the α and β2 subunits, respectively) [START_REF] Wulf | Molecular investigations of BK(Ca) channels and the modulatory beta-subunits in porcine basilar and middle cerebral arteries[END_REF], while the molecular mass of the α subunit in mammalian mitochondria is variable, depending on tissue (55-125 kDa) [START_REF] Xu | Cytoprotective role of Ca 2+ -activated K + channels in the cardiac inner mitochondrial membrane[END_REF]36]. The β2 subunit of mitoBK Ca was detected in astrocyte mitochondria [37]. However, the molecular identity (gene and protein sequences) of the mammalian mitoBK Ca channel is presently unknown. On the other hand, the molecular mass of the detected potato tuber mitoBK Ca channel α subunit (~70 kDa) is similar to the plant potassium channel protein from the plasma membrane of Arabidopsis thaliana (76 kDa) [38].

Our results demonstrate the presence of a mitoBK Ca -channel in plant mitochondria that may function as a possible novel signaling link between intramitochondrial calcium levels and the mitochondrial membrane potential. Ion channels in the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to plant cell function. At least one type of potassium channel, the mitoK ATP channel, has been described in plant mitochondria to date [START_REF] Pastore | The existence of the K + channel in plant mitochondria[END_REF][START_REF] Petrussa | Cyclosporin A induces the opening of a potassium-selective channel in higher plant mitochondria[END_REF][START_REF] Chiandussi | Modulation of a plant mitochondrial K + ATP channel and its involvement in cytochrome c release[END_REF][START_REF] Diolez | Correlations between ATP-synthesis, membrane potential and oxidation rate in plant mitochondria[END_REF]. The physiological functions of the plant mitoK ATP channel remain unclear. It may be involved in regulating mitochondrial volume [START_REF] Petrussa | Cyclosporin A induces the opening of a potassium-selective channel in higher plant mitochondria[END_REF], in programmed cell death [START_REF] Casolo | Involvement of the mitochondrial K + ATP channel in H 2 O 2 -or NO-induced programmed death of soybean suspension cell cultures[END_REF], and/or in the prevention of reactive oxygen species formation [START_REF] Pastore | The existence of the K + channel in plant mitochondria[END_REF][START_REF] Ruy | A Highly active ATP-insensitive K+ import pathway in plant mitochondria[END_REF]. The physiological role of the mitoBK Ca channel in the plant mitochondria described in this work, which seems to significantly modulate K + mitochondrial distribution, awaits exploration.

In summary, in the present study we identified and characterized a novel potassium channel of the inner membrane of potato tuber mitochondria. Pharmacological, biophysical, and molecular properties of the potato tuber mitochondrial potassium channel are similar to those of the well known mammalian plasma membrane BK Ca , including: (i) potassium selectivity and activation by calcium ions and NS1619 (a BK Ca A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. opener); (ii) inhibition by IbTx (a BK Ca blocker); (iii) biophysical properties of a large-conductance potassium channel; (iv) immunoreactivity with antibodies raised against α and β subunits of the plasma membrane BK Ca . These properties strongly suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in plant mitochondria. The functional and immunoblotting data presented herein demonstrate for the first time the presence of a mitoBK Ca channel in the inner membrane of plant mitochondria. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 09 Sep 2009 as manuscript BJ20090991 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090991

Biochemical

  Journal Immediate Publication. Published on 09 Sep 2009 as manuscript BJ20090991 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090991

Biochemical

  Journal Immediate Publication. Published on 09 Sep 2009 as manuscript BJ20090991 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090991 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 09 Sep 2009 as manuscript BJ20090991 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090991A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 09 Sep 2009 as manuscript BJ20090991 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090991

  36. Douglas, R.M., Lai, J.C.K., Bian, S., Cummins, L., Moczydlowski, E. and Haddad, G.G. (2006) The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain. Neuroscience 139, 1249-1261 37. Piwonska, M., Wilczek, E., Szewczyk, A. and Wilczyński, G.M. (2008) Diferential distribution of Ca 2+ -activated channel β4 subunit in rat brain: immunolocalization in neuronal mitochondria. Neuroscience 153, 446-60 38. Reintanz, B., Szyroki, A., Ivashikina, N., Ache, P., Godde, M., Becker, D., Palme, K. and Hedrich, R. (2002) AtKC1, a silent Arabidopsis potassium channel alpha -subunit modulates root hair K+ influx. Proc. Natl. Acad. Sci. USA 99, 4079-4084

Figure 1

 1 Figure 1 Iberiotoxin-sensitive influence of CaCl 2 on resting respiratory rate and membrane potential. Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. A, 5 mM succinate, 1.5 µM IbTx and successive doses of CaCl 2 were added as indicated. The dashed trace shows the measurement obtained in the absence of Ca 2+ . An example of three measurements (using mitochondria from three different preparations) is shown. Numbers on the traces refer to O 2 consumption rates in nmol O × min -1 × mg -1 protein or to ∆Ψ values in mV. B, Ca 2+ -induced IbTx-sensitive respiration versus CaCl 2 concentration. Inset, double reciprocal plot. C, D, effect of CaCl 2 concentration on O 2 consumption and ∆Ψ (respectively) in the presence or absence of 1.5 µM IbTx. Course of the measurements as in A, except that IbTx was added prior to addition of Ca 2+ where indicated. The data deal with three different mitochondrial preparations (± S.D). C, the percentage of control respiration in the absence of modulators (119 ± 4 nmol O × min -1 × mg -1 protein). D, the change in ∆Ψ induced by a given concentration of CaCl 2 relative to the initial state 4 ∆Ψ (216 ± 1 mV, S.D.) in the absence of modulators.

Figure 2

 2 Figure 2 Influence of NS1619, a mitoBK Ca channel activator, on resting respiratory rate and membrane potential. Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. A, 5 mM succinate, 1.5 µM IbTx, and successive doses of NS1619 were added as indicated. The dashed trace shows the measurement obtained in the absence of activator. Numbers on the traces refer to O 2 consumption rates in nmol O × min -1 × mg -1 protein or to ∆Ψ values in mV. B, NS1619-induced IbTx-sensitive respiration versus NS1619 concentration. Inset, double reciprocal plot. C, D, effect of NS1619 concentration on O 2 consumption and ∆Ψ (respectively) in the presence or absence of 1.5 µM IbTx. Course of the measurements as in A, except that IbTx was added prior to activator addition where indicated. The data deal with three different mitochondrial respirations (± S.D). C, the percentage of control respiration in the absence of modulators (122 ± 3 nmol O × min -1 × mg -1 protein). D, the change in ∆Ψ induced by a given concentration of CaCl 2 relative to the initial state 4 ∆Ψ (217 ± 1 mV, S.D.) in the absence of modulators.

Figure 3

 3 Figure 3 Influence of iberiotoxin, a mitoBK Ca channel inhibitor, on resting respiratory rate and membrane potential. Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. A, 5 mM succinate, 40 µM NS1619, and successive doses of 0.5 µM IbTx were added as indicated. The dashed trace shows the measurement in the absence of IbTx addition. Numbers on the traces refer to O 2 consumption rates in nmol O × min -1 × mg -1 protein or to ∆Ψ values in mV. B, IbTx-sensitive NS1619-induced respiration versus the inhibitor concentration. Inset, double reciprocal plot. C, D, effect of IbTx concentration on O 2 consumption and ∆Ψ (respectively) in the presence or absence of 0.8 mM CaCl 2 or 40 µM NS1619. Course of measurements as in A. The data deal with three different mitochondrial respirations (± S.D). C, the percentage of control respiration in the absence of activators and IbTx (120 ± 4 nmol O × min -1 × mg -1 protein). D, the change in ∆Ψ induced by a given modulator relative to the initial state 4 ∆Ψ (216 ± 1 mV, S.D.).

Figure 4

 4 Figure 4 Cation selectivity; influence of cations on Ca 2+ -induced IbTx-sensitive resting respiratory rate and membrane potential. Mitochondria were incubated in a standard incubation medium, except that 3 mM NaH 2 PO 4 was replaced with 3 mM TrisH 2 PO 4 . Increasing concentrations of KCl, NaCl, LiCl, CsCl, or RbCl (10-50 mM) were obtained by successive additions once a steady-state ∆Ψ had been established. The dilution effect of salt addition was taken into account. The change in ∆Ψ induced by a given chloride salt relative to the initial state 4 ∆Ψ (216 ± 1 mV, S.D.) is shown as the difference between ∆Ψ measured in the presence of 0.8 mM CaCl 2 and ∆Ψ measured in the presence of CaCl 2 and 1.5 µM IbTx.

Figure 5

 5 Figure 5 Single channel recordings of large-conductance potassium channels from potato tuber mitochondria in planar lipid bilayers. A, Schematic configuration of the cis and trans compartments used in the experiments. Reconstitution of inner mitochondrial membranes into planar lipid bilayers was performed as described under "Experimental Procedures". B, Single channel current-time recordings in 50/450 mM KCl (cis/trans) gradient solutions at different voltages. "-" indicates current during the closed state of the channel. An example of each of eight measurements is shown. C, current-voltage (I/V) characteristics of single-channel events in 50/450 mM KCl (cis/trans) gradient solutions (n = 12) at different voltages. D, probability of channel opening (P) at two different voltages, 0 and 50 mV (mean ± SEM, n = 3).

Figure 6

 6 Figure 6 Effects of Ca 2+ on the activity of large-conductance potassium channels from potato tuber mitochondria. A, an example of the single channel recordings in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV under control conditions and after addition of 300 µM Ca 2+ to the cis and trans compartments (n = 4). "-" indicates current during the closed state of the channel. B, amplitude histograms measured in the absence and presence of 300 µM Ca 2+ . All points shown in the amplitude histograms were fitted by two Gaussian distributions. The closed state corresponds to the peak at 0 pA, O -open state, C -closed state. C, Probability of channel opening (P) at 0 mV potential. The average of four different experiments (± SEM) is shown.

Figure 7

 7 Figure 7 Effects of IbTx on the activity of large-conductance potassium channels from potato tuber mitochondria. A, an example of single channel recordings in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV under control conditions and after addition of 600 nM IbTx to the cis and trans compartments. "-" indicates current during the closed state of the channel. B, amplitude histograms from a control experiment and after addition of 600 nM IbTx. All points shown in the amplitude histograms were fitted by two Gaussian distributions. The closed state corresponds to the peak at 0 pA, O -open state, Cclosed state. C, Probability of channel opening (P) at 0 mV potential. Data from eight experiments in which different concentrations of IbTx were used are presented as means ± SEM.

Figure 8

 8 Figure 8 Western blot analysis of potato tuber fractions with anti-K Ca 1.1 (A) and anti-sloβ β β β2 (B) antibodies raised against the mammalian BK Ca channel proteins in the absence or presence of a specific blocking peptide. C, Detection of H + -ATPase. Mito -mitochondria, SMP -submitochondrial particles, PM -plasma membrane-enriched fraction. Different amounts of protein were loaded into each lane (as indicated). Examples of 3-4 immunoblots (using samples from different preparations) are shown.

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© 2009 The Authors Journal compilation © 2009 Portland Press Limited

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This work was supported by the Polish Mitochondrial Network MitoNet.pl and by the Nencki Institute of Experimental Biology. Partially the study was supported by grants from the Ministry of Science and Higher Education P-N/031/2006 and 3382/B/P01/2007/33.