Vian Azzu

Nadeene Parker

Martin D Brand

High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

Keywords: hydroxynonenal, ROS, reactive oxygen species, TPMP, triphenylmethylphosphonium cation, UCP, uncoupling protein 4-hydroxynonenal (HNE), proton leak, carboxyatractylate (CAtr), rat liver mitochondria, trypsin

Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, we show that energisation (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast to the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4hydroxynonenal and/or carboxyatractylate.

INTRODUCTION

Excessive reactive oxygen species (ROS) production is deleterious to cells, and is implicated in the aetiology and/or progression of a wide variety of pathologies including cardiovascular and neurodegenerative diseases [START_REF] Dalle-Donne | Protein carbonylation, cellular dysfunction, and disease progression[END_REF][START_REF] Perry | Reactive Oxygen Species Mediate Cellular Damage in Alzheimer Disease[END_REF][START_REF] Jeremy | Reactive oxygen species, vascular disease and cardiovascular surgery[END_REF]. However, ROS also play a role in signal transduction [START_REF] Droge | Free radicals in the physiological control of cell function[END_REF], and recently Echtay et al. proposed that ROS and their downstream lipid peroxidation products like 4-hydroxynonenal (HNE) are not merely cytotoxic, but act as signals that result in mitochondrial uncoupling via the uncoupling proteins (UCPs) and adenine nucleotide translocase (ANT) [START_REF] Echtay | Superoxide activates mitochondrial uncoupling proteins[END_REF][START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]. The mitochondrial respiratory chain is a significant producer of ROS [START_REF] Mclennan | The contribution of mitochondrial respiratory complexes to the production of reactive oxygen species[END_REF][START_REF] Lenaz | The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology[END_REF], particularly at high mitochondrial membrane potential [START_REF] Korshunov | High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria[END_REF]. Echtay and others have suggested that ROS-induced mild uncoupling via UCPs/ANT effectively acts as a negative feedback loop by decreasing mitochondrial membrane potential, thereby attenuating further ROS production [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]10].

The proteins that are thought to contribute to proton leak in mitochondria belong to the mitochondrial solute carrier family [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF][START_REF] Andreyev | The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria[END_REF][START_REF] Andreyev | Carboxyatractylate inhibits the uncoupling effect of free fatty acids[END_REF][START_REF] Samartsev | Involvement of aspartate/glutamate antiporter in fatty acidinduced uncoupling of liver mitochondria[END_REF]. Despite extensive research into uncoupling pathways via the UCPs and ANT, the molecular mechanisms by which activators induce proton conductance remain contentious. For example, fatty acids have long been known to activate ANT [START_REF] Andreyev | The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria[END_REF][START_REF] Andreyev | Carboxyatractylate inhibits the uncoupling effect of free fatty acids[END_REF] and UCPs [START_REF] Locke | Fatty acids as acute regulators of the proton conductance of hamster brown-fat mitochondria[END_REF][START_REF] Wojtczak | Effect of fatty acids on energy coupling processes in mitochondria[END_REF], but there is no consensus on how this activation is mediated. The main suggested mechanisms are the flip-flop model [START_REF] Skulachev | Fatty acid circuit as a physiological mechanism of uncoupling of oxidative phosphorylation[END_REF], the cofactor model [START_REF] Winkler | Effect of fatty acids on H + transport activity of the reconstituted uncoupling protein[END_REF], and the functional competition model [START_REF] Rial | Brown-adipose-tissue mitochondria: the regulation of the 32000-Mr uncoupling protein by fatty acids and purine nucleotides[END_REF][START_REF] Shabalina | Native UCP1 displays simple competitive kinetics between the regulators purine nucleotides and fatty acids[END_REF][START_REF] Gonzalez-Barroso | The uncoupling protein UCP1 does not increase the proton conductance of the inner mitochondrial membrane by functioning as a fatty acid anion transporter[END_REF]. The flip-flop model is generally invoked to explain uncoupling of ANT by fatty acids. All three models are discussed for UCPs, and should also be considered for ANT (see [START_REF] Lou | Mitochondrial uncouplers with an extraordinary dynamic range[END_REF]). In the flip-flop model, a protonated fatty acid crosses the mitochondrial inner membrane into the matrix, dissociates, and the fatty acid anion is translocated back out of the matrix by the protein. Cycling of the fatty acid anion in its protonated and unprotonated forms results in net flux of protons into the matrix, thereby causing uncoupling. In the cofactor model, fatty acid carboxyl groups introduce a protonconducting pathway. Attempts to clarify whether activation occurs via the flip-flop model or the cofactor model have included using 'unflippable' lipids such as glucose-O-ω-palmitate. However, data from different laboratories appears to conflict (reviewed in [START_REF] Porter | A new look at UCP 1[END_REF]). In the functional competition model, fatty acids are not directly involved in the transport mechanism, but competitively overcome inhibition of a native proton conductance by ATP without displacing the nucleotide from the protein.

Our knowledge of alkenal induction of proton leak is even less well characterised [START_REF] Cannon | Uncoupling proteins: A role in protection against reactive oxygen species-or not?[END_REF]. The main proposal is that alkenals covalently modify target proteins, thereby increasing their proton conductance [START_REF] Brand | Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins[END_REF]. Numerous studies show that alkenals such as HNE form adducts with ANT [START_REF] Yan | Mitochondrial adenine nucleotide translocase is modified oxidatively during aging[END_REF][START_REF] Chen | Inhibition of adenine nucleotide translocator by lipid peroxidation products[END_REF][START_REF] Choksi | Oxidatively damaged proteins of heart mitochondrial electron transport complexes[END_REF], but this has not formally been linked to uncoupling. Others have suggested that alkenals only stimulate uncoupling if they are first converted to a fatty acid [START_REF] Shabalina | UCP1 and defense against oxidative stress. 4-Hydroxy-2-nonenal effects on brown fat mitochondria are uncoupling protein 1-independent[END_REF].

Following the finding that activation of uncoupling by HNE in skeletal muscle mitochondria requires energisation [START_REF] Parker | Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential[END_REF], and given our inability to discriminate between UCP3 and ANT using inhibitors [30], we chose to characterise the factors involved in this phenomenon using a UCP-free system. In the present study, we use the interaction of HNE with ANT in rat liver mitochondria as a model for defining the factors involved in activation of uncoupling by alkenals. The rat liver system is ideal for characterising HNE-induced uncoupling because of the abundance of ANT compared with UCPs (which simplifies data interpretation) and the availability of a tight-binding ANT inhibitor such as carboxyatractylate (CAtr) [START_REF] Klingenberg | The binding of atractylate and carboxy-atractylate to mitochondria[END_REF] (which allows ANT-specific activity to be calculated). We demonstrate that in the presence of HNE, energisation (specifically high membrane potential) is required to activate proton leak through ANT, that this uncoupling is not reversible by CAtr (in contrast to fatty acid-induced proton leak through ANT, which has been shown to be both preventable and reversible by CAtr [START_REF] Andreyev | Carboxyatractylate inhibits the uncoupling effect of free fatty acids[END_REF][START_REF] Locke | Fatty acids as acute regulators of the proton conductance of hamster brown-fat mitochondria[END_REF]), and that the proton conductance and nucleotide translocation pathways of ANT are behaviourally distinct.

EXPERIMENTAL

Animals

Female Wistar rats (Charles River Laboratories, UK) were housed at 21 ± 2°C, humidity 57 ± 5% with a 12 h light/dark cycle. Food and water were available ad libitum. Home Office Guidelines for the Care and Use of Laboratory Animals (UK) were followed.

Tissue collection and mitochondrial isolation

Liver was dissected from rats after killing by stunning and cervical dislocation. Mitochondria were prepared by the method of [START_REF] Chappell | Subcellular components: preparation and fractionation[END_REF]. The following steps were carried out at 4°C. Tissue was placed in STE buffer (250 mM sucrose, 5 mM Trizma base, 2 mM EGTA, pH 7.4), minced, homogenised, and centrifuged at 1047 g for 3 min. The supernatant was decanted and centrifuged at 11621 g for 10 min. The pellet was resuspended in STE and centrifuged at 11621 g for 10 min twice more. The final pellet was resuspended in approximately 1-1.2 ml

Respiratory control ratios

To estimate the functional quality of mitochondria, the respiratory control ratio (state 3/state 4 oligomycin ) was measured for each preparation (see below for oxygen consumption measurements). State 3 respiration was induced in the presence of 4 mM succinate by 600 µM ADP and terminated to state 4 by the addition of 1 µg/ml oligomycin. All mitochondria used had a respiratory control ratio of at least four.

Proton leak kinetics

Oxygen consumption and membrane potential were measured simultaneously at 37°C, using a Clark-type oxygen electrode and an electrode sensitive to the potential-dependent cation, triphenylmethylphosphonium (TPMP) [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF]. Mitochondria were incubated at 0.5 mg protein/ml in air-saturated KPHEB medium (120 mM KCl, 5 mM KH 2 PO 4 , 3 mM HEPES, 1 mM EGTA, pH 7.2, supplemented with 0.3% (w/v) defatted bovine serum albumin).

Additions of 5 µM rotenone (a complex I inhibitor, to prevent oxidation of endogenous NADlinked substrates), 1 µg/ml oligomycin (to prevent ATP synthesis), and 80 ng/ml nigericin (to abolish the pH gradient) were made, in the presence or absence of 2.5 µM CAtr (Calbiochem) and/or 35 µM HNE (Cayman).

The chamber was sealed, avoiding air bubbles, and the TPMP electrode was calibrated with sequential additions of 0.5 µM TPMP to a final concentration of 2.5 µM. To calculate membrane potentials, data were corrected for TPMP binding to mitochondria using a binding correction of 0.4 (µl/mg protein) -1 [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF]. Proton leak rates were calculated by multiplying respiration rates by the H + /O ratio of six for succinate oxidation. Mitochondria were energised with 4 mM succinate, followed by titration with malonate or KCN (up to 2.3 mM or 0.2 mM respectively) to vary post-energisation membrane potential and respiration rate for determination of proton leak kinetics. To vary the membrane potential during energisation, pre-additions of malonate or cyanide were made before succinate. After each run, 0.3 µM FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone) was added to release TPMP for baseline correction.

ANT-specific proton leak was calculated as total proton leak inhibitable by preaddition of 2.5 µM CAtr. CAtr may also (indirectly) prevent proton leak through UCPs [30], and UCP2 can be expressed in liver mitochondria under certain conditions [START_REF] Larrouy | Kupffer cells are a dominant site of uncoupling protein 2 expression in rat liver[END_REF][START_REF] Lee | Tumor necrosis factor increases mitochondrial oxidant production and induces expression of uncoupling protein-2 in the regenerating mice [correction of rat] liver[END_REF][START_REF] Carretero | Transformed but not normal hepatocytes express UCP2[END_REF][START_REF] Hodny | High expression of uncoupling protein 2 in foetal liver[END_REF]. However, immunoblotting of UCP2 calibrated using recombinant human UCP2 standards showed UCP2 content to be negligible, constituting 0.00022 ± 0.00003% of total mitochondrial protein (data not shown), forty-fold less than in lung and 160-fold less than in spleen [START_REF] Harper | Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria[END_REF].

Measurement of ANT content by carboxyatractylate titration

Given the high specificity and tight binding of CAtr to ANT [START_REF] Klingenberg | The binding of atractylate and carboxy-atractylate to mitochondria[END_REF], the minimum concentration of CAtr required to lower state 3 respiration rates to state 4 levels equals ANT content.

Respiration rates were measured as above, without oligomycin or nigericin. After a 5 min energisation with 4 mM succinate in the presence or absence of 35 µM HNE, excess ADP (600 µM) was added to establish state 3 respiration. This was then successively inhibited by CAtr additions (up to 3 nmol/mg mitochondrial protein) until state 4 was well established.

ANT content was calculated as the point at which the gradients in state 3 and state 4 intersected when respiration rate was plotted as a function of the amount of CAtr added.

Trypsin digestion assay

Mitochondria were treated as for proton leak assays, but to terminate succinate oxidation after 5 min, 0.29 µM myxothiazol (a complex III inhibitor) was added. The whole incubation (KPHEB medium containing 0.5 mg/ml mitochondria, 5 µM rotenone, 1 µg/ml oligomycin, 80 ng/ml nigericin, 2.5 µM TPMP, ± 35 µM HNE, ± 2.5 µM CAtr, 4mM succinate and 0.29 µM myxothiazol) was frozen (-20°C) and thawed (37°C) to rupture membranes, and subsequently treated with 0.25 mg/ml trypsin at 37°C. At various times, aliquots were treated with 0.5 mg/ml soybean trypsin inhibitor to stop trypsin digestion, and centrifuged at 16000 g for 5 min. The supernatant was removed. The mitochondrial pellet was resuspended in gel loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) bromophenol blue), boiled for 5 min and vortexed vigorously.

Immunoblotting

Proteins from the trypsin digestion assay were separated by 12% SDS-PAGE [39], transferred onto a Protran nitrocellulose membrane (Schleicher and Schuell UK ), and probed with 0.8 µg/ml of an anti-human ANT goat polyclonal IgG antibody (sc-9300, Santa Cruz Biotechnology). The secondary antibody was a horseradish peroxidase-conjugated donkey anti-goat IgG used at 0.08 µg/ml (sc-2020, Santa Cruz Biotechnology). The immunoblot was developed using a Lumigen® ECL Plus Western Blotting Detection system (Amersham Biosciences). Protein was quantified by analysing band intensities using ImageJ software (http://rsb.info.nih.gov/ij/). Membranes were stained with Gelcode Blue Stain Reagent (Pierce) to verify digestion of mitochondrial proteins by trypsin.

Chemicals

All chemicals were from Sigma-Aldrich and BDH except where otherwise stated.

Data analysis

Data are quoted as means ± SEM unless otherwise stated. Significance was tested by single factor ANOVA with Tukey's honestly significant difference post hoc analysis. Values of *P<0.05 and **P<0.01 were considered significant.

RESULTS

Time-dependent uncoupling through ANT in energised rat liver mitochondria

Carboxyatractylate is a tight-binding inhibitor of ANT [START_REF] Klingenberg | The binding of atractylate and carboxy-atractylate to mitochondria[END_REF], so the CAtr -sensitive portion of uncoupling was used as a measure of proton leak mediated by ANT. Initial measurements of proton leak kinetics revealed that ANT-mediated proton conductance increased progressively after the mitochondria were energised by addition of succinate. This is shown in Fig. 1A, where at any given driving force (membrane potential) the proton leak rate under a variety of conditions was higher when mitochondria were energised with succinate for 5 min than for 2.5 min. Proton leak under these conditions at the highest common membrane potential (137 mV) is shown in Fig. 1B. ANT-mediated proton conductance increased more prominently in the presence of 35 µM HNE in its absence (control). To show the effect of HNE at shorter times more clearly, Fig. 1C shows the analysis at the highest membrane potential common to the 2.5 min data points (175 mV). Even with only 2.5 min of energisation, proton leak through ANT was significantly greater in the presence of HNE than in the control. Subsequent experiments also using 2.5 min energisation (Figs. 2 & 3) gave proton conductances through ANT in the presence of HNE that were not significantly different from It has previously been shown that the uncoupling effect of HNE is observable even at 1 µM HNE, but that 35 µM is required in the presence of BSA [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]. Although perhaps more physiologically relevant, the effect at 1 µM would be too small to allow investigation of its characteristics, particularly in the presence of BSA to minimise fatty-acid-mediated uncoupling through ANT, therefore HNE was used at 35 µM in all our experiments.

The activation of ANT-mediated proton conductance was dependent upon the time between initiation of energisation and assay of proton leak, rather than on the total incubation time of mitochondria with or without HNE (data not shown). The effect of longer energisation times on control proton leak are dealt with in more detail in [30], which also shows that increased proton leak is an energisation-dependent effect and not a result of damage to mitochondria that are stirred for longer periods.

Although CAtr stabilises the c-conformation of ANT, which is thought to promote opening of the mitochondrial permeability transition pore [START_REF] Haworth | Control of the mitochondrial permeability transition pore by high-affinity ADP binding at the ADP/ATP translocase in permeabilized mitochondria[END_REF], our medium contained excess EGTA, which inhibits pore opening. In our experiments CAtr did not increase proton leak through ANT as would be the case with pore opening, but inhibited it, so proton leak did not involve pore opening in the results presented here.

Thus the proton conductance of ANT increases with time of energisation, particularly when HNE is present.

Membrane potential-dependent uncoupling through ANT in energised rat liver mitochondria

To determine whether membrane potential was responsible for the energisation-dependent activation of proton leak through ANT in a UCP-free model, we progressively decreased the state 4 membrane potential during energisation by pre-addition of different concentrations of the succinate dehydrogenase inhibitor, malonate (Figs. 2A-D). Analysis at the highest common potential (170 mV) shows that in the presence of HNE, proton leak through ANT decreased at higher concentrations of malonate, (Fig. 2E). This was not the case in the absence of HNE (Fig. 2E).

Since malonate additions not only lower membrane potential but also progressively oxidise the electron transport chain, we tested whether changes in redox state of the respiratory chain were responsible for the energisation-dependent activation of proton leak through ANT. We decreased the membrane potential during energisation by pre-addition of cyanide, a complex IV inhibitor that progressively reduces the electron transport chain (Figs. 3A-D). Cyanide pre-additions resulted in decreased proton conductance through ANT in the presence, but not the absence of HNE (Fig. 3E), showing that the redox state of the electron transport chain was not an important factor.

To make direct comparisons between the data in Figs. 2 and3, proton leak rates were normalised to the maximum rate on each day, then averaged. ANT-mediated proton leak rates at fixed membrane potential were then plotted as a function of the drop in state 4 membrane potential during energisation (Fig. 4). The data sets followed the same pattern, suggesting that it is indeed membrane potential that is important for HNE-induced proton leak through ANT (Fig. 4A). The loss of membrane potential did not affect the control (endogenous) proton conductance at fixed energisation duration (Fig. 4B), implying that the HNE-activated and endogenous proton leak pathways through ANT differ.

Thus the activation of proton conductance after 2.5 min through ANT caused by addition of HNE requires the presence of a high membrane potential, whilst that of the endogenous pathway does not.

Irreversibility of HNE-induced uncoupling through ANT

Fig. 5 examines the reversibility of HNE-induced activation of ANT. CAtr was either added before energisation with succinate, to prevent activation, or at 1, 3 or 5 min after energisation, to test whether activation could be reversed. Proton leak kinetics were then measured 5 min after succinate addition (Fig. 5A). Fig. 5B shows ANT-dependent proton leak rates measured at the highest common potential of 127 mV, which were calculated by subtracting the rate with CAtr added at the start of the experiment. CAtr added 3 min or 5 min after energisation with succinate did not reverse the HNE-induced activation of proton conductance through ANT, since the rates were the same as when no CAtr was added after energisation. CAtr added 1 min after energisation was partially inhibitory, presumably because it prevented further time-dependent activation by HNE rather than reversing activation that had already occurred.

Thus the time-dependent increase in proton conductance of ANT caused by addition of HNE is prevented by pre-incubation with CAtr but is not reversed by subsequent addition of CAtr after 3 min of energisation.

ANT conformation during activation of HNE-induced uncoupling through ANT

The lack of reversibility by CAtr of the effect of HNE on ANT-mediated proton leak raises two possibilities: either HNE modifies ANT conformation sufficiently to prevent CAtr binding, or CAtr still binds to ANT in the presence of HNE but no longer prevents proton leak through it. We investigated whether HNE modifies ANT conformation by using trypsin digestion (whereby the susceptibility of ANT trypsin digestion sites is affected by the conformation that ANT assumes under given conditions). Figs. 6A andB show that exogenous trypsin rapidly degrades ANT in mitochondria, and CAtr is strongly protective (presumably by binding tightly and altering ANT conformation, since Coomassie staining of membranes revealed that CAtr had no effect on trypsin's ability to digest other mitochondrial proteins (Figs. 6C &D)). Addition of HNE did not alter the susceptibility of ANT to digestion by trypsin, and did not affect the ability of CAtr to protect (Figs. 6A &B), suggesting that HNE does not modify ANT conformation sufficiently to prevent CAtr binding.

Thus treatment with HNE does not prevent CAtr from protecting ANT against degradation by trypsin, suggesting that CAtr still binds to ANT in the presence of HNE even though it no longer prevents proton leak through ANT.

No effect of HNE on state 3 respiration rate or CAtr titre.

To investigate further whether CAtr still binds to ANT in the presence of HNE, we titrated state 3 respiration with CAtr, with or without HNE pre-treatment (Fig. 7). HNE did not affect the ability of low concentrations of CAtr to inhibit state 3 respiration (Fig. 7A), suggesting that HNE did not alter binding of CAtr. This conclusion was confirmed by assaying the amount of CAtr required to fully inhibit state 3 to the state 4 rate: CAtr titre was unchanged in HNE-treated mitochondria, showing that CAtr still binds tightly and inhibits adenine nucleotide transport after pre-treatment with HNE (Fig. 7B).

Thus treatment with HNE does not prevent CAtr from binding tightly to ANT and inhibiting adenine nucleotide transport, even though treatment with HNE prevents CAtr from inhibiting proton leak through ANT. This suggests that the proton conductance and nucleotide translocation pathways in ANT are behaviourally and perhaps physically distinct.

DISCUSSION

In this study, we used ANT as a model for understanding the factors involved in HNEinduced mitochondrial uncoupling. The results may also be relevant to the activation of UCPs by HNE [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]. Our data show that energisation, by generating high membrane potential, plays an important role in the ability of HNE to activate ANT-mediated proton conductance, and that increased time at high membrane potential results in greater uncoupling. This timedependent activation appears to be irreversible over the short term by subsequent CAtr addition. Our data also imply that the proton conductance pathway is distinct from the nucleotide translocation pathway in ANT.

We find that after 2.5 min energisation, the HNE-induced pathway behaves differently from the endogenous pathway in being membrane potential-dependent. In light of [30], this may be because the endogenous pathway has not been sufficiently activated at 2.5 min in liver, or it may be explained by tissue-specific differences. In liver, time at high membrane potential in the presence of HNE results in a striking increase in proton conductance, which may be interpreted in two ways. Firstly HNE may slowly form covalent adducts with ANT, progressively converting ANT to a form with high proton conductance, or secondly, HNE metabolism may progressively form fatty acids [41], which then activate proton conductance [START_REF] Shabalina | UCP1 and defense against oxidative stress. 4-Hydroxy-2-nonenal effects on brown fat mitochondria are uncoupling protein 1-independent[END_REF]. The lack of reversibility of HNE-induced proton conductance by CAtr (Fig. 5) is unlike fatty acid-mediated activation, which is known to be both prevented and reversed by CAtr [START_REF] Andreyev | The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria[END_REF][START_REF] Andreyev | Carboxyatractylate inhibits the uncoupling effect of free fatty acids[END_REF]; this shows that the effect of HNE is not mediated indirectly by fatty acids. Indeed, it implies that HNE modifies ANT permanently, perhaps by formation of covalent adducts [START_REF] Yan | Mitochondrial adenine nucleotide translocase is modified oxidatively during aging[END_REF][START_REF] Chen | Inhibition of adenine nucleotide translocator by lipid peroxidation products[END_REF][START_REF] Choksi | Oxidatively damaged proteins of heart mitochondrial electron transport complexes[END_REF]. HNE is known to form Michael adducts with sulphydryl groups and lysine or histidine residues of proteins [42]. We therefore propose that high membrane potential influences the conformation of ANT or its lipid milieu [START_REF] Beyer | ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31 P nuclear magnetic resonance[END_REF][START_REF] Hoffmann | The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants[END_REF], allowing HNE to interact with ANT and activate ANT-mediated proton conductance. Conformational changes may involve a transition of ANT from the c-state to the m-state, which unmasks ANT's sulphydryl groups [START_REF] Aquila | The reactivity of -SH groups in the ADP/ATP carrier isolated from beef heart mitochondria[END_REF] and lysine residues [START_REF] Klingenberg | Is there a binding center in the ADP, ATP carrier for substrate and inhibitors? Amino acid reagents and the mechanism of the ADP, ATP translocator[END_REF][START_REF] Block | Chemical modifications of atractyloside and bongkrekic acid binding sites of the mitochondrial adenine nucleotide carrier. Are there distinct binding sites?[END_REF][START_REF] Klingenberg | Molecular aspects of the adenine nucleotide carrier from mitochondria[END_REF], rendering them susceptible to attack by HNE. Work by Klingenberg and colleagues on purified and reconstituted ANT shows that the carrier is protected from trypsin digestion by CAtr addition, but rendered susceptible by bongkrekate addition [49].

Their ANT digestion profiles are consistent with those that we see in mitochondria that are treated (or not) with CAtr. Furthermore, Vignais and colleagues report that the uncoupler FCCP prevents alkylation of ANT sulphydryl groups, presumably by preventing a c-state to m-state transition of the carrier [START_REF] Vignais | Effect of SH reagents on atractyloside binding to mitochondria and ADP translocation. Potentiation by ADP and its prevention by uncoupler FCCP[END_REF]. In much the same way, decreasing membrane potential during energisation (Fig. 4) might prevent HNE interaction with these groups. Similarly, preincubation with CAtr would fix the c-state and prevent HNE adduct formation. Klingenberg et al. show that pre-treatment with bongkrekate induces the m-state and prevents binding of both atractylate and CAtr, but when added subsequently it only removes atractylate [START_REF] Klingenberg | The binding of atractylate and carboxy-atractylate to mitochondria[END_REF]. In contrast, our finding that CAtr can still bind to ANT after HNE treatment indicates that HNE does not behave like bongkrekate to permanently stabilise the m- conformation, but allows the carrier to revert to the c-state. Also, in contrast to the Vignais study in which sulphydryl group alkylation prevented nucleotide translocation and atractylate binding [START_REF] Vignais | Effect of SH reagents on atractyloside binding to mitochondria and ADP translocation. Potentiation by ADP and its prevention by uncoupler FCCP[END_REF], and the Chen study [START_REF] Chen | Inhibition of adenine nucleotide translocator by lipid peroxidation products[END_REF] in which HNE inhibited the nucleotide translocation activity of ANT by sulphydryl group alkylation, HNE affected neither parameter in our system (Fig. 7). It is likely that the extent of sulphydryl group modification in our system is considerably less than in [START_REF] Vignais | Effect of SH reagents on atractyloside binding to mitochondria and ADP translocation. Potentiation by ADP and its prevention by uncoupler FCCP[END_REF] and [START_REF] Chen | Inhibition of adenine nucleotide translocator by lipid peroxidation products[END_REF], which used a potent alkylating agent (Nethylmaleimide) and millimolar concentrations of HNE, respectively.

The simplest model to explain our observations (Fig. 8) is that high membrane potential favours the m-state of ANT, which is required for HNE to react. Once HNE has reacted, proton translocation is permanently switched on and insensitive to later CAtr addition, but the carrier still translocates nucleotides, cycles between the c-state and the mstate, and can be trapped in the c-state by CAtr, preventing further nucleotide transport but permitting proton transport.

An alternative hypothesis is that ANT is required for activation of uncoupling, but the HNE-induced proton conductance pathway does not subsequently require ANT. This may explain why pre-addition of CAtr prevents proton leak induction, whereas its addition after HNE makes no difference to proton leak even though it can still inhibit nucleotide translocation by the carrier.

Biochemical

  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080321 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080321 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080321 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080321 S t a g e 2 ( a ) P O S T -P R I N T of STE and stored on ice for up to 6 h. Protein concentration was determined by the biuret method.

Biochemical

  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080321 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080321 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

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Fig. 1C .

 1C Fig. 1C.

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  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080321 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080321 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

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Biochemical

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Biochemical

  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080321 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080321 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1 Proton leak kinetics in rat liver mitochondria: energisation-dependent activation (A) Rat liver mitochondria incubated in KPHEB medium plus 5 µM rotenone, 1 µg/ml oligomycin and 80 ng/ml nigericin ± 2.5 µM CAtr ± 35 µM HNE were energised with 4 mM succinate for 2.5 min (black points) or 5 min (grey points). To determine proton leak kinetics, oxygen consumption and membrane potential were measured as succinate oxidation was progressively inhibited by titration with malonate (up to 2.3 mM). (B) Proton leak rates through ANT at the highest potential common to all data sets (thick dotted line in (A)). Data are means ± SEM of at least five independent experiments. *P<0.05 and **P<0.01 compared to 2.5 min. (C) Proton leak rates through ANT at the highest common potential at 2.5 min of energisation (fine dotted line in (A)).Data are means ± SEM of 11 independent experiments. *P<0.05 compared to control.

Figure 2

 2 Figure 2 Effect of malonate pre-addition on ANT-mediated proton leak Rat liver mitochondria incubated in KPHEB plus 5 µM rotenone, 1 µg/ml oligomycin and 80 ng/ml nigericin ± 2.5 µM CAtr ± 35 µM HNE were energised with 4 mM succinate after pre-addition of different concentrations of malonate: (A) none added, (B) 0.15 mM, (C) 0.3 mM, (D) 0.6 mM. After 2.5 min of energisation, proton leak kinetics were determined by malonate titration (up to 2.3 mM). Data are means ± SEM of four independent experiments. (E) Proton leak through ANT at the highest common potential. Data are calculated as each condition minus its 'plus CAtr' condition for each independent experiment and subsequently averaged to give means ± SEM of four independent experiments. **P<0.01 compared to #. (F) Experiment time course. Time zero is addition of 0.5 mg/ml mitochondria to KPHEB medium.

Figure 3 Figure 4

 34 Figure 3 Effect of cyanide pre-addition on ANT-mediated proton leak Rat liver mitochondria incubated in KPHEB plus 5 µM rotenone, 1 µg/ml oligomycin and 80 ng/ml nigericin ± 2.5 µM CAtr ± 35 µM HNE were energised with 4 mM succinate after pre-addition of different concentrations of cyanide: (A) none added, (B) 30 µM, (C) 45 µM, (D) 60 µM. After 2.5 min of energisation, proton leak kinetics were determined by cyanide titration (up to 0.2 mM). Data are means ± SEM of four independent experiments. (E) Proton leak through ANT at the highest common potential. Data are calculated as each condition minus its 'plus CAtr' condition for each independent experiment and subsequently averaged to give means ± SEM of four independent experiments. **P<0.01 compared to #. (F) Experiment time course. Time zero is addition of 0.5 mg/ml mitochondria to KPHEB medium.

Figure 5

 5 Figure 5 Effect of CAtr on HNE-induced ANT activation (A) Rat liver mitochondria incubated in KPHEB plus 5 µM rotenone, 1 µg/ml oligomycin and 80 ng/ml nigericin ± 35 µM HNE were energised with 4 mM succinate. Where indicated, 2.5 µM CAtr was added at the start of the experiment (CAtr before En), or 1 min, 3 min or 5 min after succinate addition (CAtr after En). After 5 min of energisation ("En"), proton leak kinetics were determined by malonate titration (up to 2.3 mM). Data are means ± SEM of four independent experiments. (B) Proton leak rates through ANT at the highest common potential. Data are calculated as each condition minus its 'plus CAtr (and before HNE)' condition for each independent experiment and subsequently averaged to give means ± SEM of four independent experiments. For HNE data, **P<0.01 compared to #.

Figure 6

 6 Figure 6 Effect of CAtr and HNE on ANT conformation as probed by digestion with trypsin Rat liver mitochondria were treated as for proton leak experiments; where indicated, 2.5 µM CAtr and 35 µM HNE were added. CAtr was added at the start of the experiment (CAtr and CAtr bef. HNE) or after HNE, 5 min after addition of succinate (CAtr aft. HNE). At the point where malonate titration would be carried out, mitochondria were inhibited with myxothiazol, and subsequently frozen-thawed to rupture membranes. Thawed mitochondria in KPHEB medium were treated with 0.25 mg/ml trypsin at 37°C. At 0, 20 and 60 min after trypsin addition, aliquots were treated with 0.5 mg/ml trypsin inhibitor. Mitochondria were pelleted and proteins were separated by 12% SDS-PAGE, then immunoblotted for ANT. (A) Typical immunoblot of trypsin digestion experiment. Molecular weight markers (kDa) are shown on the left of two typical immunoblots of samples from different incubation conditions. Times (0, 20 or 60 min) after trypsin addition are indicated. (B)Trypsin digestion profiles for different conditions. Data are means ± SEM of four independent

  Figure 7 CAtr titre of respiration (A) Rat liver mitochondria incubated in KPHEB plus 5 µM rotenone ± 35 µM HNE were energised with 4 mM succinate for 5 min, then state 3 respiration was achieved by addition of 600 µM ADP. State 3 was titrated by successive additions of CAtr to bring mitochondria into a CAtr -inhibited state 4. (B) Amount of CAtr required to fully titrate state 3 to state 4, calculated from the intersection of linear regressions as shown in (A). Data are means ± SEM of four independent experiments. Data sets were not significantly different.

Figure 8

 8 Figure 8 Scheme of proposed ANT conformation under different conditions At high membrane potential (∆ψ), the c-like state (A) transitions towards an m-like state (B) thereby exposing reactive sulphydryl groups. If HNE is present, the m-like state can slowly be modified by adduct formation to a form with a higher proton conductance (C). ANT-HNE can then cycle between m-like (C) and c-like (D) states, allowing normal nucleotide transport. The c-like states (A, D) may still be trypsin-sensitive as de-energised mitochondria are degraded with similar profiles as shown in Fig. 6 (unpublished observations). CAtr binding induces a conformational change in ANT (A CAtr , D CAtr ), which protects it from trypsin digestion and prevents adenine nucleotide translocation. The fixed c-state (A CAtr ) does not react with HNE but can accommodate pre-reacted HNE (D CAtr ), in which case it retains high proton conductance. For further details see Results and Discussion.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

Support from the Medical Research Council, the Wellcome Trust (grants 065326/Z/01/Z and 066750/B/01/Z) (NP, MDB), and the School of Clinical Medicine, University of Cambridge, UK (VA) is gratefully acknowledged.


Anja Spielvogel

Helen Findon

Herbert N Arst

Lidia Araújo-Bazán

Patricia Hernández-Ortíz

Ulf Stahl

Vera Meyer

Dr Eduardo A Espeso
email: eespeso@cib.csic.es

Two zinc finger transcription

Keywords: AFP, antifungal protein, CDD, calcineurin docking domain, CDRE, calcineurin-dependent response element, DTT, ditiotreitol, GFP, green fluorescent protein, ORF, open reading frame, WMM, watch minimal medium Aspergillus nidulans, nuclear localisation, calcium signalling, cation homeostasis, cell wall integrity, transcription factors GFP

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Introduction

Rapid adaptation to different environmental conditions, including appropriate adaptive responses to various cations, is crucial for the survival and proliferation of microorganisms. The Ca 2+ /calcineurin/Crz1p signalling pathway is necessary for adaptation to diverse environmental conditions including high cation levels and alkaline pH, and considerable characterization of it has been achieved in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Torulaspora delbrueckii and Candida albicans [START_REF] Hirayama | Zinc finger protein Prz1 regulates Ca2+ but not Cl-homeostasis in fission yeast. Identification of distinct branches of calcineurin signaling pathway in fission yeast[END_REF][START_REF] Hernandez-Lopez | Regulation of salt tolerance by Torulaspora delbrueckii calcineurin target Crz1p[END_REF][START_REF] Cyert | Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress[END_REF][START_REF] Karababa | CRZ1, a target of the calcineurin pathway in Candida albicans[END_REF]. Sudden exposure of S. cerevisiae to these stress conditions induces a transient increase in cytosolic Ca 2+ levels which triggers activation of the phosphatase calcineurin through binding of the Ca 2+ -binding protein calmodulin. S. cerevisiae calcineurin, like that of mammals is composed of both catalytic and regulatory subunits [START_REF] Cyert | Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase[END_REF]. Activated calcineurin mediates dephosphorylation of the cytoplasmically localised transcription factor Crz1p, resulting in its translocation to the nucleus ( [START_REF] Viladevall | Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae[END_REF]; reviewed in [START_REF] Cyert | Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress[END_REF]).

The nuclear localised zinc finger Crz1p transcription factor then activates expression of genes whose promoters contain physiologically relevant calcineurin-dependent response elements (CDREs) [START_REF] Yoshimoto | Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae[END_REF] 5´-GNGGC(G/T)CA-3, and up-regulates expression of various target genes, including some encoding cation transporters acting at the plasma membrane or other membranous organelles [START_REF] Matheos | Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae[END_REF][START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF][START_REF] Stathopoulos | Calcineurin acts through the CRZ1/TCN1encoded transcription factor to regulate gene expression in yeast[END_REF]. Previously we found that alkaline pH-induced up-regulation of the Aspergillus giganteus afp gene encoding the antifungal protein can be prevented by FK506, an inhibitor of calcineurin [START_REF] Meyer | Alkaline pH-induced up-regulation of the afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus is not mediated by the transcription factor PacC: possible involvement of calcineurin[END_REF]. To pursue afp regulation in the context of cation adaptation and homeostasis, we have chosen to investigate calcium-mediated signalling in Aspergillus nidulans, an experimentally more amenable species than A. giganteus.

Calcium-mediated signalling has been less investigated in filamentous fungi than in yeasts although some notable work was done on calcineurin (reviewed in [START_REF] Stie | Calcineurin Regulation in Fungi and Beyond[END_REF]) which has been revisited recently [START_REF] Soriani | Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA[END_REF]. It was initially reported that the A. nidulans gene encoding the calcineurin catalytic subunit is essential and required for cell cycle progression [14;15]. However, very recently an A. nidulans ΔcnaA, deleted for the calcineurin catalytic subunit, mutant strain was found to be viable and display a phenotype similar to that observed for the corresponding Aspergillus fumigatus (ΔcalA) mutant strain, which has severe defects in growth extension, branching and conidiophore development [START_REF] Soriani | Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA[END_REF]. In addition to having abnormal morphogenesis, the A. fumigatus ΔcalA mutant is also impaired in virulence [16;17]. In Aspergillus oryzae calcineurin is required for adaptation to elevated temperature, high Na + levels and alkalinisation [START_REF] Juvvadi | Functional analysis of the calcineurin-encoding gene cnaA from Aspergillus oryzae: evidence for its putative role in stress adaptation[END_REF]. Crz1p orthologues have been recently characterised in the filamentous fungi A. fumigatus and Botrytis cinerea, where they play major roles in tolerance to certain cations [13;19].

Another transcription factor important for cation adaptation and homeostasis is SltA. An A. nidulans mutation, designated sltA1, resulting in toxicity by high levels of Na + and K + was described a number of years ago [START_REF] Spathas | A salt sensitive mutation on chromosome VI of Aspergillus nidulans[END_REF]. Subsequently, cloning of the sltA gene by complementation of the sltA1 mutation showed that it encodes a protein containing three Cys 2 His 2 -type zinc fingers and exhibiting significant similarity to the Ace1 transcriptional repressor of cellulase and xylanase genes in Hypocrea jecorinia (Trichoderma reesei) [21]. Although O'Neil et al (2002) showed that the sltA1 mutation results from a premature stop codon just downstream of the third zinc finger of the predicted protein, they did not investigate the role of the SltA protein in cation tolerance, nor did they determine whether sltA1 has a null phenotype.

Here we characterise aspects of the role of the A. nidulans transcription factors CrzA, a homologue of S. cerevisiae Crz1p, along with SltA, which has no identifiable S. cerevisiae homologue, but, like CrzA, is involved in cation adaptation and homeostasis. We show that both CrzA and SltA are capable of binding DNA and that SltA acts positively on transcription of the Ena1p-like sodium pump enaA and negatively on transcription of the putative vacuolar

Experimental

Strains, growth conditions, molecular techniques and phenotype analysis

A. nidulans strains used are listed in Table 1. Escherichia coli strains DH5α (Gibco BRL) and XL10-Gold® (Stratagene) were used as hosts for the maintenance of the plasmids listed in Supplementary Table 1. General cloning procedures in E. coli were followed. Standard growth media for A. nidulans, genetic techniques and phenotype testing followed those described previously ( [START_REF] Tilburn | The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid-and alkaline-expressed genes by ambient pH[END_REF] and references therein). Gene symbols are described in [START_REF] Clutterbuck | Aspergillus nidulans. In Genetic maps. Locus maps of complex genomes[END_REF]. Reversion alleles of sltA1 were selected in a UV-mutagenised strain of genotype halA24 inoB2 sltA1 as enabling growth on appropriately supplemented complete medium containing (final concentration) 1 M NaH 2 PO 4 , and their phenotypes were determined in a selection of outcrossed strains. A. nidulans transformation followed ( [START_REF] Tilburn | The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid-and alkaline-expressed genes by ambient pH[END_REF] and references therein). A. nidulans total RNA and genomic DNA preparation as well as Northern and Southern analyses followed standard procedures.

Identification of the crzA gene

The CrzA homologue in the A. nidulans genome was identified by Blastp searches using the S. cerevisiae Crz1p sequence. On the basis of sequence conservation and apparently similar function, AN5726 was named crzA. The position of the predicted intron within the crzA gene was verified by RT-PCR and is in agreement with GenBank entry AB259568. Strain J734 was grown in liquid minimal medium and exposed for two hours to 1 M NaCl after which total RNA was obtained. Total RNA treated with DNase I (FPLC pure, Amersham Biosciences) was used as template for the RT-PCR reaction. First strand synthesis used primer CrzA-9 rev (Supplementary Table 2) and ArrayScript ™ Reverse Transcriptase (Ambion). The following PCR performed with primers CrzA-8 fw and CrzA-9 rev yielded full length crzA cDNA which was cloned into plasmid pGEM-T-Easy (Promega) giving plasmid pGEM-T-CrzAcDNA. The insert was subsequently sequenced using primer walking.

Deletion of crzA

The crzA deletion cassette was constructed as gene replacement cassette comprising the pyr-4 gene of Neurospora crassa as selection marker flanked by 1.5 kb of 5' and 3' untranslated regions of the crzA gene. The 5' untranslated region of crzA was amplified by PCR using genomic DNA from strain J734 and primers crznulo-1 and crznulo-2, which introduced a NotI and a BamHI restriction site, respectively. The resulting PCR fragment was digested with NotI and BamHI and inserted into pBluescriptSK(+) (Stratagene) giving plasmid pBSKpromCrzA. The pyr-4 selection marker obtained from plasmid pFB6 [START_REF] Tilburn | The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid-and alkaline-expressed genes by ambient pH[END_REF] by BglII digestion was inserted into BamHI-linearized pBSKpromCrzA yielding plasmid pBSKpromCrzApyr4-fw. Correct orientation of the pyr-4 gene was verified by PCR and restriction analysis. The 3' untranslated region of crzA was amplified by PCR using genomic DNA from strain J734 and primers crznulo-3 and crznulo-4. The resulting PCR fragment was inserted into pGEM-T-Easy and excised using EcoRI digestion. The EcoRI fragment was inserted in the correct orientation into pBSKpromCrzApyr4-fw via the EcoRI restriction site. The final plasmid harbouring the 6.2 kb crzA deletion cassette was named pBSKdeltaCrzA. This cassette was amplified by PCR using primers crzAnulo1 and crzAnulo-4 and used to transform A. nidulans strain MAD1425. Pyrimidine prototrophic transformants were screened for the crzA deletion by Southern and PCR analysis and strain BER02 was selected for further work.

Deletion of sltA

A cassette comprising the A. fumigatus riboB gene fused to the upstream and downstream untranslated regions (UTRs) of sltA was used for transformation to delete the sltA coding region. A 1.5kb 5'-UTR region of sltA was amplified using primers sltnul1 and sltnul2, which included a NotI and a BamHI restriction site, respectively. The PCR fragment was digested with NotI and BamHI and cloned into the same restriction sites in the pBluescriptSK(+) (Stratagene) plasmid to give pBS-SltA5'. A 1.5kb 3'-UTR region of sltA was amplified using primers sltnul3 (inserting an EcoRI restriction site) and sltnul4 (carrying a XhoI site). The PCR fragment was digested with EcoRI and XhoI, and ligated to pBS-SltA5' to give pBS-SltA5'3'. The EcoRI 1.9 kb fragment containing the A. fumigatus riboB coding region plus 5' and 3' UTRs was obtained from plasmid p1548, and ligated to plasmid pBS-SltA5'3' digested with EcoRI to give pBSdeltaSltA. pBSdeltaSltA was digested with NotI and XhoI to yield the deletion cassette. The sltA null strain was made using MAD1427 as recipient strain selecting for transformants able to grow in the absence of riboflavin. Riboflavin prototrophs were analysed by Southern blot and one transformant carrying the desired insertion was used for further work.

GFP tagging of CrzA

Construction of an A. nidulans strain expressing CrzA::GFP followed the methodology described in [START_REF] Yang | Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans[END_REF], taking advantage of recently available strains deficient in heterologous recombination [START_REF] Nayak | A versatile and efficient gene-targeting system for Aspergillus nidulans[END_REF]. 1.5 kb from the most 3' end of the gene and the 3'-UTR region were amplified with primers pairs CrzA1/CrzA2 and CrzA3/CrzA4, respectively, (see Supplementary Table 2) and fused in a 3-way PCR with a fragment, amplified with primers CrzA-GFP1 and CrzA-GFP2, containing the gfp gene together with the selectable pyrG gene of A. fumigatus [START_REF] Yang | Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans[END_REF]. The fusion cassette was transformed into MAD1732 following the protocol described in ( [START_REF] Tilburn | The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid-and alkaline-expressed genes by ambient pH[END_REF] and references therein). Pyrimidine prototrophs were selected and homokaryotic transformants were analysed by Southern blot for single copy integration of the gene replacement cassette.

Gene expression analyses

Steady state mRNA levels of the Aspergillus nidulans ENA1-like genes were detected using specific, PCR-amplified, genome probes: For AN6642 a probe of 1357 bp using primers An6642rev and An6642fw covering 40% of the ORF was used For AN1628 a probe of 948 bp using primers An1628rev and An1628fw covering 29% of the ORF was used. For AN10982 a probe of 1295 bp using primers An10982rev and An10982fw, and covering 37% of the ORF was used. To detect AN0471 (designated vcxA), a 926 bp fragment using primers An0471rev and An0721fw, covering 65% of the ORF, was synthesised. For chsB detection (AN2523), a fragment of 719 bp was amplified using primers An2523rev and An2523fw, covering 30% of the ORF. Actin transcript detection used as probe the fifth exon, amplifying the genomic DNA with primers Anid-AcnA and Anid-AcnB. For oligonucleotide sequences see Supplementary Table 2. Northern analyses used total RNA extracted from wild type and mutant strains grown in minimal medium (MMA) adjusted to pH 6.5, containing 1% glucose and 10 mM NH 4 + [as the (+)-tartrate] as main carbon and nitrogen sources, respectively. For investigating the various cation effects, after 18 hrs of culture at 37ºC the indicated additions and pH alterations were made with further incubation as specified in the figure legends. Quantification of enaA and acnA transcription was done exposing the filters to a Phosphorimager screen (Molecular Dynamics) and developing with a FLA-5100 Reader (Fujifilm Global). Band intensities were measured using Multi-Gauge V3.0 (Fujifilm Global) software.

Construction of GST::CrzA and GST:: SltA fusion proteins

The plasmid pGEX-CrzA123(469-603) was constructed to express a GST fusion protein containing the complete zinc-finger region of CrzA (aa 469 -603). The zinc-finger region of crzA was amplified by PCR using genomic DNA of strain J734 as template and primers CrzA-ZF-fw-BamHI and CrzA-ZF-rev-EcoRI. Both oligonucleotides introduced the restriction sites for BamHI and EcoRI that allowed in-frame ligation of the PCR fragment into pGEX-2T (Amersham Biosciences). The final plasmid was named pGEX-CrzA(469-603). The plasmid expressing a GST::SltA(381 -549) fusion protein was constructed by cloning a PCR amplicon obtained from J734 genomic DNA with oligonucleotides SltA-ZF-fw-BamHI and SltA-ZF-rev-EcoRI (amplifying the entire zinc finger region of the sltA gene and introducing BamHI and EcoRI sites) into pGEX-2T. The resulting plasmid was named pGEX-SltA(381-549). Fidelity and correct orientation of the inserts were verified by sequencing. Fusion proteins were expressed in E. coli DH1 and purified by glutathione affinity chromatography according to the manufacturer's instructions (Amersham Biosciences).

Electromobility shift assays

Synthetic double-stranded oligonucleotides were generated as described in [START_REF] Orejas | Activation of the Aspergillus PacC transcription factor in response to alkaline ambient pH requires proteolysis of the carboxy-terminal moiety[END_REF]. A. giganteus sites afp-CDRE-5 and afp-SLTA-1 were obtained annealing oligonucletides CDRE-5 with CDRE-5(-) and SltA-1 with SltA-1(-), respectively. T. reesei probes 2C and 2C4 were obtained annealing oligonucleotides Ace1-2C-1 with Ace1-2C-2 and Ace1-2C4-1 with Ace1-2C4-2, respectively. For the A. nidulans chsB CDRE-1 site, oligonucleotides Chs-CDRE-1fw and Chs-CDRE-1rev, were annealed (for oligonucleotide sequences see Supplementary Table 2). Synthetic probes were labelled by end-filling using Klenow polymerase in the presence of a mixture containing dGTP and [α-32 P]dCTP. Binding reactions were performed as described previously [START_REF] Orejas | Activation of the Aspergillus PacC transcription factor in response to alkaline ambient pH requires proteolysis of the carboxy-terminal moiety[END_REF]. The binding reaction mixture (final volume 20 μl) contained binding buffer (25 mM HEPES-KOH, 50 mM KCl, 5 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, 20% glycerol; pH 7.9), 1 ng labelled double-stranded oligonucleotides, 3 μg non-specific competitor [poly(dI-dC) or poly(dA-dT)] and variable amounts of proteins, as purified GST or GST::CrzA(469-603) fusion, or as E. coli protein extracts for GST::SltA(381-549). Various amounts, as indicated in figures or legends, of unlabelled double-stranded oligonucleotides as specific competitors, were added to the reaction mixtures before the addition of protein. Reaction mixtures were kept on ice for 30 min and DNA and DNA-protein complexes were separated on native 5% poly-acrylamide gels. Gels were dried and autoradiographed.

AFP susceptibility testing

Determination of the sensitivity of A. nidulans strains to the antifungal protein AFP followed the protocol of [START_REF] Theis | The antifungal protein from Aspergillus giganteus causes membrane permeabilization[END_REF].

Microscopy

Conidia of A. nidulans strains were grown in appropriately supplemented liquid minimal medium placed on cover slips. Germlings adhering to cover slips were observed using an Axioskop 50 (Zeiss) equipped with a Camedia Digital Camera C-4000 Zoom (Olympus, Hamburg). Light images were captured with a 40 x objective. Images were processed using Adobe Photoshop 6.0 (Adobe Systems Inc.). Observations of Aspergillus fluorescent strains were done in ''watch minimal medium'' (WMM) as described in [START_REF] Penalva | Tracing the endocytic pathway of Aspergillus nidulans with FM4-64[END_REF]. The Aspergillus strain expressing CrzA::GFP was cultured on the surface of glass bottom petri dishes immersed in 2.5 ml of appropriately supplemented WMM at 25ºC during 16 h. Media were replaced by fresh WMM containing either with or without 10 mM CaCl 2 and the green fluorescence was observed using a Leica DMI6000b inverted microscope, equipped with a GFP specific filter (Ex.470nm; Em. 525nm), and recorded with an Orca-ER camera (Hamamatsu) driven by Metamorph (Universal Image Co.) CrzA and Crz1p share 66% identity within the putative DNA binding domain which comprises three zinc fingers, the first two of which conform to the Cys 2 -His 2 consensus [(Y/F)XCX 2-4 CX 3 (Y/F)X 5 LX 2 HX 3-4 H] (Figure 1A). This degree of sequence conservation suggests that CrzA and Crz1p might bind the same DNA target sequence. The third finger of Crz1p and its homologues, including CrzA, appears to have an atypical Cys 2 -His-Cys structure with unusual spacing (Figure 1A) and is currently under study. The 730-residue CrzA protein, in common with its homologues, contains the DNA binding domain in its Cterminal moiety. CrzA contains two overlapping putative calcineurin docking domains (CDD) (Figure 1B) whereas Crz1p which is activated by dephosphorylation by the calcineurin phosphatase is thought to contain a single specific recognition sequence PxIxIT [START_REF] Aramburu | Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT[END_REF][START_REF] Roy | A conserved docking site modulates substrate affinity for calcineurin, signaling output, and in vivo function[END_REF][START_REF] Boustany | Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site[END_REF]. Putative Crz1p orthologues are widely distributed amongst the fungi with sequence conservation notably high amongst filamentous fungi (Figure 1C and Supplementary Figure 1). The crzA transcript appears to be expressed at a very low level by northern analysis but is increased somewhat by exposure to 100 mM Ca 2+ for periods of 5-30 min (data not shown). In contrast, no increase in A.fumigatus crzA transcript levels was reported in response to 10 or 60 min exposure to 200 mM Ca 2+ [START_REF] Soriani | Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA[END_REF]. The very modest increase in crzA transcript levels in response to Ca 2+ suggests that regulation might be mainly post-transcriptional, particularly in view of the effect of Ca 2+ on CrzA localisation (see below).

The phenotype of a crzA null mutation.

To assess the physiological role of CrzA, a strain deleted for crzA was constructed by gene replacement using the Neurospora crassa pyr-4 gene as selectable marker, as described in Experimental. The original transformant and outcrossed ΔcrzA strains are, like the corresponding deletion mutants of A. fumigatus and B. cinerea [13;19], strongly inhibited by elevated Ca 2+ concentrations and alkaline pH (Figure 2), thus resembling the behaviour of crz1 mutants [9;32] but much more extreme.

Strains deleted for crzA have delayed germination in minimal medium in which Ca 2+ is present only as a trace contaminant (Figure 3A). The presence of 100 mM Ca 2+ causes hyphal hyperbranching in ΔcrzA strains (Figure 3B). Similar morphological abnormalities have been noted for the corresponding A. fumigatus and B. cinerea deletion mutants [13;19].

Interestingly ΔcrzA strains show significant resistance to the toxicity of the antifungal protein AFP (Figure 3C), recently shown to inhibit chitin biosynthesis, very probably as a result of inhibition of chitin synthases of classes III and V [START_REF] Hagen | The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi[END_REF]. In contrast, ΔcrzA strains show slight hypersensitivities to other cell wall perturbing compounds such as caspofungin and congo red (data not shown), further suggesting a role for CrzA in determining cell wall structure. Cell wall abnormalities have also been reported for the orthologous B. cinerea crz1 deletion mutant [START_REF] Schumacher | Calcineurin-responsive zinc finger transcription factor CRZ1 of Botrytis cinerea is required for growth, development, and full virulence on bean plants[END_REF]. The similarities in phenotype between null crzA and null chsB A. nidulans strains, which lack a class III chitin synthase [34;35], suggested that chsB might be under CrzA control. Figure 3D shows that whereas transfer to medium containing 100 mM Ca 2+ elevates transcript levels of chsB in wild type, it fails to do so in a ΔcrzA strain, indicating that up-regulation of chsB in response to Ca 2+ is CrzA-dependent. In agreement, the chsB promoter contains two putative CDRE sites (data not shown) conforming to the Crz1p binding site consensus [START_REF] Yoshimoto | Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae[END_REF]. In contrast to the effect of Ca 2+ , 300 mM Li + appears to have no effect on chsB transcript levels (Figure 3D).

Another target of CrzA regulation is AN0471.3, denoted vcxA AN0471 , which we identified as the closest A. nidulans homologue of S. cerevisiae VCX1 using tBlastn and Blastp searches. In S. cerevisiae the vacuolar Ca 2+ /H + exchanger Vcx1p mediates import of excess cytosolic Ca 2+ into the vacuole [START_REF] Pozos | The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers[END_REF]. VCX1 transcription is negatively regulated by calcineurin in a Crz1p-independent manner [8;37]. Figure 4A shows that vcxA AN0471 transcript levels are increased by 100 mM Ca 2+ in a CrzA-dependent manner. As a large number of calcium-regulated S. cerevisiae genes show a greater transcriptional response at an early time after alkalinisation provokes a transient rise in cytosolic calcium levels than at a later time [START_REF] Viladevall | Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae[END_REF], vcxA AN0471 transcript levels were monitored at both 30 and 60 min following calcium exposure. Indeed, steady-state transcript levels were lower at the later time point (Figure 4A). Expression of VCX1 homologues in A. fumigatus and B. cinerea is also dependent on the respective CRZ1 orthologues [13;19].

Subcellular localisation of CrzA.

To analyse the subcellular distribution of CrzA, a mutant crzA allele expressing a Cterminally GFP-tagged CrzA was constructed by gene replacement using previously described procedures [24;25;38]. The protein CrzA::GFP encoded by the tagged allele appears to be fully functional as no Ca 2+ sensitivity or growth impairment at alkaline pH is observable (data not shown). In the absence of Ca 2+ addition and any form of stress, CrzA::GFP is exclusively cytosolic, apparently excluded from nuclei (Figure 5). Upon addition of CaCl 2 to a final concentration of 10 mM, CrzA rapidly accumulates in nuclei, eventually depleting the cytoplasm of fluorescence (Figure 5 and data not shown). Thus, as previously described for the CrzA orthologue Crz1p ([6;31;39] and reviewed in [START_REF] Cyert | Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress[END_REF]), CrzA appears to be subject to an active nuclear export and import machinery, with an increase of cytosolic Ca 2+ concentration eliciting its nuclear import (Figure 5). Presumably, as in S. cerevisiae [START_REF] Boustany | Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site[END_REF], Ca 2+ activation of calcineurin results in dephosphorylation of CrzA and consequent loss of nuclear export capability. A. fumigatus CrzA also localises to the nucleus in response to Ca 2+ [START_REF] Soriani | Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA[END_REF] as does B. cinerea CRZ1 when expressed heterologously in S. cerevisiae [START_REF] Schumacher | Calcineurin-responsive zinc finger transcription factor CRZ1 of Botrytis cinerea is required for growth, development, and full virulence on bean plants[END_REF].

Further characterization of sltA mutations.

In view of the sensitivity of ΔcrzA strains to toxicity of the cation Ca 2+ , further characterization of mutations in the sltA gene where the sltA1 allele confers sensitivity to the cations Na + and K + [20;21;40] seemed warranted. Although SltA homologues are widely distributed in filamentous fungi, we have been unable to detect any in searches of yeast genomes. The sltA gene encodes a 698-residue protein containing three Cys 2 -His 2 zinc fingers and the sltA1 mutation changes tryptophan codon 502, the second codon following that of the last histidine of finger 3, to a stop codon (Supplementary Figure 2A) [21].

To determine the phenotype of a null sltA allele and to see whether sltA1 has the null phenotype a deletion allele was constructed by gene replacement using the Aspergillus fumigatus riboB gene as selectable maker (Experimental; Supplementary Figure 2B). We have detected no phenotypic differences between ΔsltA and sltA1, suggesting that at least some portion of the C-terminal 197 residues is essential to SltA function (data not shown). As seen in Figure 2, the absence of functional SltA results in elevated sensitivity to Li + , Cs + and Mg 2+ in addition to Na + and K + , inability to grow at alkaline pH and hypersensitivity to neomycin toxicity. In plate tests ΔcrzA ΔsltA double mutants have phenotypes which are the sum of the single mutant phenotypes (Figure 2).

Although overall conservation of the C-terminal sequence beyond the third zinc finger of SltA is low among putative homologues, tryptophan-502 and the residues immediately downstream from it are highly conserved, suggesting this region might be required for full functionality (Supplementary Figure 2A). To test this possibility, revertants of a sltA1 strain were obtained, after ultraviolet mutagenesis, as allowing growth in the presence of 1 M NaH 2 PO 4 . As well as reversion in codon 502 to the wild type tryptophan codon, partially functional revertants with codon 502 encoding arginine, leucine, phenylalanine, lysine or cysteine were also obtained (Table 2). Unsurprisingly, the reversion to phenylalanine appears to give the greatest degree of functionality. These reversion data indicate that tryptophan-502 is itself an important residue and that one or more additional important residues is/are located C-terminal to it.

SltA acts negatively in the control of vcxA AN0471 and positively in the control of enaA AN6642

The ΔsltA mutant showed an elevated response to Ca 2+ in vcxA AN0471 transcript levels at both 30 and 60 min and the response is more pronounced at the latter time point (Figure 4B; data not shown for 30 min). However, ΔcrzA is epistatic to ΔsltA in the control of vcxA AN0471 as in the ΔcrzA ΔsltA double mutant there is no response to Ca 2+ in vcxA AN0471 transcript levels (Figure 4B), correlating with the fact that Ca 2+ inhibition of ΔcrzA ΔsltA double mutants is very similar to that of ΔcrzA single mutants (Figure 2). Using S. cerevisiae Ena1p, Ena2p and Ena5p in Blastp and tBlastn searches of the A. nidulans genome (http://www.broad.mit.edu/annotation/genome/aspergillus_group/Blast.html) three loci whose predicted protein products are closely related to the Ena P-type ATPase sodium pumps, AN6642.3, designated enaA [START_REF] Han | Osmotic stress-coupled maintenance of polar growth in Aspergillus nidulans[END_REF], AN1628.3 and AN10982.3, were identified (data not shown). We have designated these genes enaA AN6642 , enaB AN1628 and enaC AN10982 . In S. cerevisiae the Ena transporters are involved in the salt stress response and are required for growth at alkaline pH [42;43].

Expression of enaA AN6642 requires a combination of elevated [Na + ] and alkaline pH (Figure 6). We have been unable to detect transcripts for enaB AN1628 and enaC AN10982 in the conditions or genetic backgrounds shown in Figure 6. Neither 300 mM Li + nor 100 mM Ca 2+ is sufficient to induce expression of any of the ena genes (data not shown). Figure 6 shows that SltA plays a significant, positive-acting role in enaA AN6642 expression. If CrzA plays a role, it is much more marginal.

CrzA and SltA are both DNA-binding proteins.

The putative DNA-binding domains, containing the entire zinc finger regions, residues 469-603 for CrzA and residues 381-549 for SltA were expressed in E. coli as glutathione-Stransferase (GST) fusion proteins. Although purification of GST::CrzA(469-603) using glutathione affinity chromatography was feasible, we did not succeed in purifying GST::SltA(381-549) despite trying several elution conditions (data not shown). Consequently bacterial protein crude extract were used in DNA binding studies.

To characterise the DNA binding ability of GST::CrzA(469-603) probes conforming to the S. cerevisiae Crz1p consensus site CDRE [START_REF] Yoshimoto | Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae[END_REF] were synthesised (Figure 7A). Two putative CDRE sites were identified in the A. nidulans chsB promoter and five such sites were identified in the Aspergillus giganteus afp promoter, previously suggested to be under the control of the Ca 2+ -calcineurin signalling pathway [START_REF] Meyer | Alkaline pH-induced up-regulation of the afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus is not mediated by the transcription factor PacC: possible involvement of calcineurin[END_REF]. CrzA binds to CDRE-1 from the chsB promoter and to CDRE-5 from the afp promoter (Figures 7B andC), confirming that binding sequences for CrzA and Crz1p are very similar or identical.

Because of the sequence similarity of SltA to Trichoderma reesei ACEI in the zinc finger region ([21]), the ability of GST::SltA(381-549) to bind to the previously described 2C and mutant 2C4 probes [START_REF] Saloheimo | Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei[END_REF] was tested. In similarity to ACEI binding, SltA binds to the 2C but not the 2C4 probe (Figure 7D). SltA also binds to the SLTA-1 site from the afp promoter, which conforms to the 5'-AGGCA ACEI consensus (Figures 7A, D andE).

As the trinucleotide 5'-GGC is common to sequences bound by CrzA and SltA, we tested whether either of the fusion proteins might bind to a site bound by the other. However, we found no evidence for binding of SltA to afp CDRE-5 (Figure 7D) or of CrzA to afp SLTA-1 (Figure 7E).

Discussion

The two putative transcription factors investigated here are both involved in preventing toxicity of certain cations. In the absence of functional SltA, A. nidulans is abnormally sensitive to toxicity by Na + , K + , Li + Cs + and Mg 2+ but not by Ca 2+ whereas the absence of CrzA results in toxicity of Ca 2+ . Transcript levels of the putative vacuolar Ca 2+ /H + exchanger gene vcxA in response to high Ca 2+ levels provide a possible explanation for the Ca 2+ toxicity to ΔcrzA strains and the lack of Ca 2+ toxicity in ΔsltA strains. Whereas ΔsltA strains show an elevated response to Ca 2+ in vcxA transcript levels, putatively allowing greater vacuolar storage and thus detoxification of Ca 2+ , ΔcrzA strains apparently show no Ca 2+ response at all, putatively resulting in elevated cytosolic Ca 2+ levels. The VcxA homologue Vcx1p of S. cerevisiae mediates the vacuolar import of excess cytosolic Ca 2+ [START_REF] Pozos | The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers[END_REF]. One possible factor in cation toxicity in sltA null strains is the reduction in induced levels of the putative cation exporter EnaA AN6642 . The homologous S. cerevisiae plasma membrane exporter Ena1p enables a detoxification response to Na + , Li + and alkalinity [42;43].

The presence of three putative SltA/ACEI binding sites 5' to the enaA coding region and one such site 5' to the vcxA coding region is consistent with SltA regulation of transcript levels of these genes. Five putative CDRE sites lie upstream of the vcxA coding region consistent with CrzA transcriptional regulation of vcxA. One CDRE site lies upstream of the enaA coding region but, in view of the lack of solid evidence that CrzA regulates enaA, its significance, if any, is unclear. In any case, further work will be necessary to determine any physiological relevance of these 5' sites.

This work has highlighted significant differences between A. nidulans and S. cerevisiae. S. cerevisiae lacks an identifiable SltA homologue and whereas S. cerevisiae Crz1p is not involved in VCX1 expression but plays a crucial role in ENA1 expression [START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF], A. nidulans CrzA plays a crucial role in vcxA expression (in similarity to findings in A. fumigatus and B. cinerea) but is hardly involved, if at all, in enaA AN6642 regulation. The role of CrzA in calcium-induced chsB expression finds a parallel in Candida albicans where calcium stimulation of promoter activity for four different chitinase genes is Crz1p-dependent [START_REF] Munro | The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans[END_REF].

A further point of interest is that SltA appears to play both a positive (in enaA AN6642 regulation) and a negative (in vcxA regulation) regulatory role with the caveat that we have not yet shown that SltA acts directly on the regulation of the genes in question. The PacC transcription factor, mediating control of gene expression by ambient pH in A. nidulans acts as both an activator and a repressor (reviewed in [START_REF] Penalva | Regulation of gene expression by ambient pH in filamentous fungi and yeasts[END_REF]) but its S. cerevisiae homologue Rim101p exerts positive regulation by repressing transcription of genes encoding other transcriptional repressors [START_REF] Lamb | The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae[END_REF]. Finally we note that at least three transcription factors are necessary for growth of A. nidulans at alkaline pH: PacC (reviewed in [START_REF] Penalva | Regulation of gene expression by ambient pH in filamentous fungi and yeasts[END_REF]), and, as established here, SltA and CrzA. In the case of SltA, at least one pertinent factor is its role in enaA AN6642 expression. Spores were point-inoculated on agar plates containing minimal medium (MMA, pH 6.5; 1% glucose as carbon source) supplemented with different salts, as indicated, or neomycin (625 ng/ml). Alkaline pH plate (pH ~8) contained 100 mM Na 2 HPO 4 . 200 mM NaCl is a Na + control for the alkaline pH medium. Plates were incubated for two days at 37°C. wt: wild-type (strain HHF17a), ΔcrzA (strain HH17d), ΔsltA (strain HH17e), ΔcrzA ΔsltA (strain HH17f). the ΔcrzA allele were cultivated in liquid MMA (pH 6.5) for 18 h at 37ºC after which CaCl 2 was added to a final concentration 100 mM. After additional cultivation for 30 or 60 min, total RNA was isolated, blotted and hybridised with a chsB probe. Methylene blue-stained 18S rRNA served as loading control. HFF17a (wt), HHF17d (ΔcrzA) HHF17e (ΔsltA) and HHF17f (ΔcrzA ΔsltA). The growth and treatment protocol followed that described for Figure 3. carrying ΔcrzA, ΔsltA, both or neither were cultivated in liquid MMA (pH 6.5) for 18 h at 37ºC after which NaCl was added (final concentration 1 M) and/or the pH of the culture broth was adjusted to pH 8 (using NaOH). After an additional cultivation for 60 min, total RNA was isolated, blotted and hybridised with enaA and acnA (actin) probes. The following strains were used in this experiment: HHF17a (wt), HHF17d (ΔcrzA) HHF17e (ΔsltA) and HHF17f (ΔcrzA ΔsltA). Levels of enaA transcripts were determined and expressed relative to those of the actin mRNAs. [START_REF] Stathopoulos | Calcineurin acts through the CRZ1/TCN1encoded transcription factor to regulate gene expression in yeast[END_REF] and [START_REF] Yoshimoto | Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae[END_REF], respectively. The core sequences of these binding sites are indicated. Probes CDRE-5 and SLTA-1 are synthetic sites from the antifungal protein promoter (afp) of A. giganteus. Probe 2C is one of the ACEI binding sites found in the T. reeseii cbh1 promoter and 2C4 is a 2C mutant probe as described in [START_REF] Saloheimo | Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei[END_REF]. Probe CDRE-1 is a synthetic site from the A. nidulans chsB promoter. (B) CrzA binding to an afp-CDRE-5 probe. (C) Binding of CrzA to a CDRE present in the chsB promoter. Binding to this site is subject to competition by the same unlabelled probe or the afp-CDRE-5 probe. For (B) and (C), numbers at the top of the gel refer to ng of purified protein used in the assay. (D) Binding of SltA to the 2C probe. 1.5 μg of E. coli extract expressing GST::SltA was used in each reaction. Affinity and specificity of binding is shown by competition with 1000x (300ng) of unlabelled probes SLTA-1, CDRE-5, 2C and 2C4. (E) Specific recognition of afp-SLTA-1 probe by SltA. Binding of SltA is shown using different amounts (μg) of E. coli crude extract expressing GST::SltA. Purified GST::CrzA does not bind to this probe.

Tables and Figures

  Biochemical Journal Immediate Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T Results Identification of the crzA gene.The putative A. nidulans homologue of S. cerevisiae CRZ1, AN5726.3 denoted crzA, was identified by Blastp searches using the full-length Crz1p protein sequence. The in silico predicted cDNA sequence of crzA was verified by sequencing a RT-PCR product obtained from total RNA.

Biochemical

  Journal Immediate Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1 Sequence analysis of CrzA. (A) Multiple alignment of the zinc finger domain of predicted CrzA homologues from yeast and filamentous fungi. Shown is the zinc finger region containing three C 2 H 2 zinc fingers (zf), each indicated by an open bar. Two paralogues were found in Candida albicans (Calb(1) and (2)). Protein alignments were done using the program ClustalX [48]. (B) Alignment of a selected number of Crz homologues showing the putative calcineurin docking domain (CDD). Open rectangles above the alignment indicate the limits of the two putative CDDs in CrzA orthologues from filamentous fungi, which are compared at the right with the CDD present in Crz1p and the consensus established from the CDDs present in the NFAT protein [29]. (C) Phylogenetic tree obtained from the full alignment of the Crz1p/CrzA homologues. The phylogenetic tree was built using MEGA 3.0 [49]. The bootstrap value obtained after 10,000 replicates is shown on top of each branch. The scale bar indicates relative evolutionary distance. For panels A and B, Residue shading follows BLOSUM32 similarities. Protein sequences are as follows: Anid, Aspergillus nidulans BAE94327; Aory, Aspergillus oryzae BAE57003; Afum, Aspergillus fumigatus EAL88401; Mgri, Magnaporthe grisea XP_359644; Ncra, Neurospora crassa EAA32849; Cglo, Chaetomium globosum EAQ88414; Gzea, Gibberella zeae EAA68022; Cimm, Coccidioides immitis EAS33001; Pnod, Phaeosphaeria nodorum EAT87393; Dhan, Debaryomyces hanseni, CAG84727; Tdel, Torulaspora delbrueckii AAZ04388; Agos, Ashbya gossypii AAS51722; Scer, Saccharomyces cerevisiae CAA95889; Klac, Kluyveromyces lactis CAG99429; Ylip, Yarrowia lipolytica CAG80473; Calb(1), Candida albicans EAK97605; Calb(2), Candida albicans EAK93473; Cgla, Candida glabrata CAG62620; Spom, Schizosaccharomyces pombe Q09838.

Figure 2

 2 Figure 2 Phenotypic analysis of A. nidulans strains defective for CrzA and/or SltA function. Spores were point-inoculated on agar plates containing minimal medium (MMA, pH 6.5; 1% glucose as carbon source) supplemented with different salts, as indicated, or neomycin (625 ng/ml). Alkaline pH plate (pH ~8) contained 100 mM Na 2 HPO 4 . 200 mM NaCl is a Na + control for the alkaline pH medium. Plates were incubated for two days at

Figure 3

 3 Figure 3 Effect of deletion of crzA on germination, morphology, AFP sensitivity and expression of the chsB chitin synthase gene. (A) Conidia from strains MAD1425 (wt) and BER02 (ΔcrzA) were grown on cover slips at 37°C for 6 or 12 h. (B) Strains HFF17a (wt), HHF17d (ΔcrzA) HHF17e (ΔsltA) and HHF17f (ΔcrzA, ΔsltA) were grown on MMA agar plates in the presence or absence of 100 mM CaCl 2 . Microscope photographs, using 10 x magnification, were taken of the colony edges. (C) 1000 conidia obtained from strains MAD1425 (wt) and BER02 (ΔcrzA) were cultivated at 28ºC in 150 μl complete medium (YPG) in the presence of different AFP concentrations. After 48 h, the optical densities were measured. Mean values of triplicate experiments are shown. (D) chsB transcript levels were determined in strains MAD1425 (wt) and BER02 (ΔcrzA) treated for 60 min with 100 mM CaCl 2 or LiCl. The untreated controls are indicated by (-). Strains carrying either wt crzA + or

Figure 4

 4 Figure 4 Effects of various genotypes and elevated Ca 2+ levels on vcxA expression. (A) vcxA transcript levels were determined after 30 and 60 min in the presence (+) or absence (-) of CaCl 2 . wt: strain MAD1425, ΔcrzA: strain BER02. (B) Expression of vcxA was determined at 30 and 60 min (only shown for the latter) after Ca 2+ addition in the following strains:

Figure 5

 5 Figure 5 Subcellular distribution of CrzA. An A. nidulans strain, MAD2055, expressing CrzA::GFP was grown in WMM at 25ºC in glass bottom petri dishes for microscopy. Mycelia were transferred to fresh medium with or without 10 mM CaCl 2 and photographed after 10 min. incubation at 25ºC. Nuclei devoid of fluorescence, indicated by arrows, are visible in cells growing in medium without added Ca 2+ . In the presence of Ca 2+ , the nuclei become strongly fluorescent. Images at the bottom show in more detail the fluorescence distribution within the hyphae under both growth conditions. The bar scales indicate 5 μm.

Figure 6

 6 Figure 6 Effects of various treatments and genotypes on enaA expression. Strains

Figure 7 DNA

 7 Figure7DNA binding capabilities and specificities of CrzA and SltA. (A). Alignment of Crz1p/CrzA and ACEI/SltA DNA binding sites. CDRE consensus sequences 1 and 2 correspond to those described in[START_REF] Stathopoulos | Calcineurin acts through the CRZ1/TCN1encoded transcription factor to regulate gene expression in yeast[END_REF] and[START_REF] Yoshimoto | Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae[END_REF], respectively. The core sequences of these binding sites are indicated. Probes CDRE-5 and SLTA-1 are synthetic sites from the antifungal protein promoter (afp) of A. giganteus. Probe 2C is one of the ACEI binding sites found in the T. reeseii cbh1 promoter and 2C4 is a 2C mutant probe as described in[START_REF] Saloheimo | Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei[END_REF]. Probe CDRE-1 is a synthetic site from the A. nidulans chsB promoter. (B) CrzA binding to an afp-CDRE-5 probe. (C) Binding of CrzA to a CDRE present in the chsB promoter. Binding to this site is subject to competition by the same unlabelled probe or the afp-CDRE-5 probe. For (B) and (C), numbers at the top of the gel refer to ng of purified protein used in the assay. (D) Binding of SltA to the 2C probe. 1.5 μg of E. coli extract expressing GST::SltA was used in each reaction. Affinity and specificity of binding is shown by competition with 1000x (300ng) of unlabelled probes SLTA-1, CDRE-5, 2C and 2C4. (E) Specific recognition of afp-SLTA-1 probe by SltA. Binding of SltA is shown using different amounts (μg) of E. coli crude extract expressing GST::SltA. Purified GST::CrzA does not bind to this probe.

Biochemical

  Journal Immediate Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T

  Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T

TABLE 1 :

 1 A. nidulans strains used in this work

	Strain	Genotype

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

back to wild-type TTG. Biochemical Journal Immediate Publication. Published on 12 May 2008 as manuscript BJ20080344 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080344 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We thank Barbara Walewska, Susanne Engelhardt, Tatiana Munera Huertas and Elena Reoyo for technical assistance and Berl Oakley for plasmid p1548 carrying the A. fumigatus riboB gene. We are grateful to the Biotechnology and Biological Sciences Research Council for support through grant BB/D52178/1 to H.N.A. and to the Spanish Ministerio de Educación y Ciencia (D.G.I.C.Y.T) for support through grant BFU2006-04185 to E.A.E.. L.A.-B. is a predoctoral fellow of the P.F.P.U. of the Spanish Ministerio de Educación y Ciencia.