Stefan H Millson

Cara K Vaughan

Chao Zhai

Maruf M U Ali

Barry Panaretou

Peter W Piper

Laurence H Pearl

Chrisostomos Prodromou
email: chris.prodromou@icr.ac.uk

Chaperone ligand-discrimination by the TPR-Domain Protein Tah1

Keywords: Hsp90, Hsp70, Tah1, Sti1/Hop/p60, ATPase activity, TPR-domains

Ligand discrimination by tetratricopeptide-domains

Abbreviations: tetratricopeptide, TPR; ITC, isothermal titration calorimetry Tah1 has been identified as a tetratricopeptide (TPR)-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino acid sequence alignments suggest that Tah1 is most similar to the TPR2b-domain of Hop which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1 which is consistent with the architecture of the TPR2b-domain. We find that Tah1 is specific for Hsp90, able to bind tightly the yeast Hsp90, and the human Hsp90 and Hsp90 proteins, but not the yeast Hsp70, Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD-motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD-motif (Ssa1). We also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

INTRODUCTION

Heat shock protein 90 (Hsp90) molecular chaperone plays a central role in the maturation and activation of key signalling proteins (reviewed in [START_REF] Picard | Heat-shock protein 90, a chaperone for folding and regulation[END_REF][START_REF] Pratt | Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery[END_REF]) that include steroid hormone receptors [START_REF] Joab | Common non-hormone binding component in non-transformed chick oviduct receptors of four steroid hormones[END_REF][START_REF] Picard | Chaperoning steroid hormone action[END_REF] and protein kinases [START_REF] Cutforth | Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila[END_REF][START_REF] Aligue | A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90[END_REF][START_REF] Dai | Physical interaction of mammalian CDC37 with CDK4[END_REF][START_REF] Oppermann | A cellular protein that associates with the transforming protein of Rous sarcoma virus is also a heat-shock protein[END_REF][START_REF] Sepehrnia | Heat shock protein 84 forms a complex with mutant p53 protein predominantly within a cytoplasmic compartment of the cell[END_REF] amongst others [START_REF] Picard | Heat-shock protein 90, a chaperone for folding and regulation[END_REF][START_REF] Pratt | Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery[END_REF][START_REF] Wilhelmsson | The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein[END_REF]11]. An up-to-date comprehensive list can be found at http://www.picard.ch/downloads/downloads.htm The activation of such proteins (client proteins) is regulated by the association of specific cochaperones that complex with Hsp90, at varying stages of the chaperone cycle [START_REF] Prodromou | Structure and functional relationships of Hsp90[END_REF][START_REF] Pearl | Structure and mechanism of the hsp90 molecular chaperone machinery[END_REF]. A number of these co-chaperones play an apparent role in recruiting client proteins to the Hsp90 chaperone complex. In the case for steroid-hormone receptors it appears that the receptor is complexed with the teratricopeptide (TPR)-domain containing co-chaperone Hop/Sti1 and Hsp70. Binding of Hop/Sti1 to the conserved MEEVD-motif at the extreme Cterminal end of Hsp90 delivers the steroid hormone receptor to Hsp90 (reviewed in [START_REF] Picard | Chaperoning steroid hormone action[END_REF][START_REF] Smith | Tetratricopeptide repeat cochaperones in steroid receptor complexes[END_REF]). Protein kinases on the other hand are complexed with Cdc37/p50 (reviewed in [15]) that binds the N-terminal domain of Hsp90 [16]. In Sacharomyces cerevisiae, Tah1 (YCR060W), consisting of 111 amino acid residues, was identified as an Hsp90 interacting co-chaperone in three independent studies [START_REF] Huh | Global analysis of protein localization in budding yeast[END_REF][18][19]. Tah1 is thought to associate with Pih1 and together complex with the essential DNA helicases, Rvb1 and Rvb2 that are key components of the chromatin remodelling complexes Ino80 and SWR-C [19]. Tah1 contains a single TPRdomain with at least two TPR-motifs [19]. Numerous crystallographic structures of TPR-domain containing proteins have been solved [20][21][22][23][24][25]. These reveal a common motif that is composed of a degenerate 34 amino acid sequence that forms an antiparallel alpha-helical hairpin, which in turn cluster to form a domain with a grooved surface that acts as a peptide-binding site. TPR-domains mediate protein-protein interactions in numerous protein complexes [26][27][28]. The question of ligand discrimination by TPR-domains has been addressed [29][30][31]. TPR-domain proteins such as Hip have been implicated in the regulation of the ATPase activity of Hsp70, while Hop/Sti1 has been shown to inhibit the ATPase activity of Hsp90, to activate Ssa1, but to have no affect on the vertebrate Hsp70 [32][33][34][35][36]. The TPR-domains of Hsp70 and Hsp90 cochaperones interact with the extreme C-terminal amino acid sequences of their respective chaperone [34,[37][38][39][40][41], which share a common motif (EEVD). Residues immediately upstream of the conserved EEVD-motif of Hsp70 and Hsp90 achieve selective binding to different 25]. In contrast, the co-chaperone CHIP, involved in the ubiquitylation of chaperone-bound client proteins [42,43], binds to both Hsp70 and Hsp90 by associating with the MEEVD-(Hsp90) or the IEEVD-motif (human Hsp70) and avoiding further interaction with upstream amino acid residues. This is achieved by a hydrophobic pocket that accommodates the methionine residue (M in MEEVD) from Hsp90 and the isoleucine residue (I in IEEVD) of human Hsp70 and in doing so twists the upstream peptide, which differs between the two chaperones, and hoists it clear of the TPR-domain groove [24]. In contrast, structural information showed that for Hop/Sti1 the N-terminal TPR-domain (TPR1) interacts with Hsp70, whereas the central TPR-domain (TPR2a) binds to Hsp90 [21]. However, the situation might be more complex in that mutations in the third TPR-domain (TPR2b) disrupt association with both Hsp70 and Hsp90 [39]. A high affinity ligand for the TPR2b-domain has not been found [21,29]. In this paper we show that the TPR-domain of Tah1 most closely resembles the TPR2bdomain of Hop/Sti1 and address whether Tah1 is an Hsp90 specific co-chaperone or whether it can also associate specifically with Ssa1. We show that Tah1 is specific for Hsp90 and that ligand discrimination is achieved by favourable binding of the methionine residue of the conserved MEEVD-motif of Hsp90, and by positively discriminating against the first valine of the VEEVD-motif of Ssa1. We also characterise the oligomeric state of Tah1 and investigate its ability to regulate the ATPase activity of Hsp90.

MATERIALS AND METHODS

Sequence alignments

The amino acid sequences for Tah1, Sti1 and Hop were obtained from Genbank. The accession numbers were, Human Hop, NM_006819; Saccharomyces cerevisiae Sti1, Z74935 and S. cerevisiae Tah1, NC_001135. The sequence alignment was generated using Clustal W

[44] 'on-line' at the European Bioinformatic Institute, which is part of the European Molecular Biology Laboratory (EMBL-EBI).

Protein production and Hsp90 ATPase activity assays

Expression and purification of wild type and mutant forms of His-tagged yeast Hsp90, Histagged Hsp90 , mouse His-prescission tagged CHIP and His-Tagged C-terminal domain of Sti1 (cSti1) was previously described [24,35,45]. The yeast Tah1, the human Hsp90 and the C-terminal domain of yeast Hsp90 were cloned into pRSETA as NheI-EcoRI, NheI-PstI and NheI-HindIII DNA fragments, respectively, and expressed as His-Prescission tagged proteins. The yeast Ssa1 gene was cloned into pRSETA as an NheI-HindIII DNA fragment and expressed as a His-tagged fusion. The same protein purification procedure as used for yeast Hsp90 was employed to purify the C-terminal domain of Hsp90, Hsp90 , Ssa1 and Tah1. PreScission cleavage of the His-tags was carried out overnight at 4 o C, as described by the manufacturer (GE Healthcare). The cleaved sample was then passed through a 1 ml GST-Hi Trap column, equilibrated in 20 mM Tris pH 7.5, 140 mM NaCl and 2.7 mM KCl, to remove GST-tagged prescission. The flow through was collected, desalted and then subjected to Talon-affinity chromatography to remove uncleaved His-Prescission Tah1 protein. The Talon-resin flow through was then concentrated and subjected to Superdx 75-or 200-HR gelfiltration chromatography, as appropriate. All purified proteins were dialysed against 20 mM Tris-HCl pH 7.5 containing 1 mM EDTA and 1 mM DTT and then concentrated using Vivaspin concentrators (Sartorius) with an appropriate molecular weight cut-off. The ATPase assays were performed in triplicate as previously described [45,46], using 2 µM yeast Hsp90 and either 20 µM Tah1 or 30 uM geldanamycin. Protein concentration was determined by using the extinction coefficient of the protein (wild type and mutant yeast Hsp90, 54,050; cleaved C-terminal domain of yeast Hsp90,18,260;cleaved Hsp90 ,59,625;Hsp90 58,135;Ssa1,20,045;cleaved CHIP,29,380;cSti1,34,480 and cleaved Tah1 10,295).

Isothermal titration calorimetry: stoichiometry and K d determination.

Heat of interaction was measured on a MSC system (Microcal) with a cell volume of 1.458 ml. For Tah1 and CHIP interactions, 9-11 aliquots of 27 µl of 300 µM Tah1 or CHIP were injected into 30 µM of yeast Hsp90, yeast Hsp90 M705V, human Hsp90 , human Hsp90 or yeast Hsp90 C-terminal domain at 4 o C (Tah1 experiments) or 30 o C (CHIP experiments) in 20 mM Tris-HCl pH 8.0 containing 1 mM EDTA and 5 mM NaCl. For peptide interactions, either 9-11 aliquots of 27 µl of 300 µM peptide or 14-15 aliquots of 400 µM peptide were injected into 30 µM Tah1 at 4 o C in 20 mM Tris-HCl pH 8.0 containing 1 mM EDTA and 5 mM NaCl. For cSti1 interactions 11 aliquots of 27 µl of 300 µM cSti1 were injected into 30 µM of yeast Hsp90 or yeast Hsp90 M705V at 30 o C in 20 mM Tris-HCl pH 8.0 containing 1 mM EDTA and 5 mM NaCl. Heat of dilution was determined in a separate experiment by diluting protein or peptide into buffer, and the corrected data fitted using a nonlinear least square curve-fitting algorithm (Microcal Origin) with three floating variables: stoichiometry, binding constant and change in enthalpy of interaction. For the Ssa1-Tah1 and Tah1-VEEVD peptide experiment the stoichiometry was fixed at N = 1. All peptides were accurately weighed on an analytical balance and dissolved in dialysis buffer. The column was run at 0.5 ml min -1 and was calibrated using gel-filtration standards from Bio-Rad (bovine thyroglobulin, Mr 670,000; bovine -globulin, Mr 158,000; chicken ovalbumin, Mr 44,000; horse myoglobin, Mr 17,000 andvitamin B12, Mr 1,350) andGE Healthcare (blue dextran 2,000; bovine albumin, Mr 67,500; chicken ovalbumin, Mr 43,000; bovine chymotrypsinogen A, Mr 25,700 and bovine ribonuclease A, Mr 13,700). The relative molecular weight of Tah1 was determined by logarithmic interpolation.

Analytical ultracentrifugation

PreScission cleaved Tah1 was dialyzed against 20 mM Tris pH 7.5 containing 1 mM EDTA and 5 mM NaCl. Sedimentation equilibrium analysis was performed at 20ºC in a Beckman XL-I Analytical Ultracentrifuge, following a Standard Operating Procedure SE_IO_07.1 at sample concentrations of 7.0 and 0.5 mg ml -1 (547 and 39, µM, respectively). After initial scans at 3000 rpm to check for the presence of any large aggregates, a final rotor speed of 22,000 rpm was selected. A 12 mm optical path length centerpiece was used in the cell. Scans were taken every 60 minutes and data logged to disk using the Beckman software. After 24 hours, no further change could be detected in the scans, and the run was terminated. The data were analysed by three methods using the INVEQ-, the recently derived Mfit-and a local (OS-X-profit) version of the widely used NONLIN-algorithm. Ka values were converted to Kd values by simple reciprocation.

RESULTS

Tah1 selectively binds Hsp90

Tah1 has been identified as a TPR-domain protein that interacts with Hsp90. Our sequence alignments suggest that Tah1 is most similar to the TPR2b-domain of Hop/Sti1 (Supplementary Figure 1), which influences the binding of Hop to both Hsp70 and Hsp90 [39]. In order to investigate the ability of Tah1 to bind both Ssa1 and Hsp90 we used isothermal titration calorimetry (ITC) to determine the extent of any interaction. Figure 1a shows that Tah1 binds to the full-length yeast Hsp90 (K d = 0.78 ± 0.06 µM) with a stoichiometry of 1:1, while Figure 1b shows that the binding site is solely located in the Cterminal domain of Hsp90 (K d = 0.32 ± 0.03 µM; Table 1 summarises all ITC data presented in this paper). Tah1 was also shown to be able to interact with the human Hsp90 (K d = 0.33 ± 0.03 µM) and Hsp90 (K d = 0.34 ± 0.07 µM) paralogs with a similar affinity (Figure 1c,d). However, its interaction with the yeast Ssa1 was significantly weaker (K d = 16 ± 2.5 µM; Figure 1e), where the strength of the interaction is reflected in the shape of the binding curve for which a steeper gradient in the observed curve indicates tighter binding (compare Figure 1a and2e), and suggests that Tah1 is an Hsp90 specific co-chaperone.

Comparison of MEEVD-peptide bind-ing to Tah1 and TPR2a

In the TPR1-VEEVD and TPR2a-MEEVD complex most of the direct hydrogen bonded interactions with bound-peptide involve the main-chain and are therefore sequence independent. Both complexes have a highly conserved two-carboxylate clamp. In the TPR2a complex the terminal main-chain carboxylate of MEEVD is hydrogen bonded to Lys229, Asn233 and Asn264. These residues are also conserved in Tah1 (Lys8, Asn12 and Asn43, respectively; see Supplementary Figure 1 and Figure 2). However, in contrast, residues that interact with the side-chain of the terminal aspartate, which is bound by Lys301 and Gln298 in TPR2a, are not conserved in Tah1 (Thr70 and His73, respectively). Consequently, if a two-carboxylate clamp exists in the peptide-bound Tah1 complex it is clearly different to that seen in TPR2a. Other electrostatic interactions include a tetrahedrally coordinated water molecule that interacts with the carboxylate side-chain of the terminal aspartate, and with the main-chain carbonyl of Thr263 (Ser42 in Tah1), the guanidinium group of Arg305 (Arg77 in Tah1) and the side-chain carbonyl of Asn264 (Asn43 in Tah1). The side-chain amide of Asn264 is also involved in main-chain interactions with the backbone amide of the terminal aspartate residue, while the guanidinium group of Arg305 also contacts the main-chain carbonyl and amide (via a water molecule) of Glu-2 (S -6 RMEEVD 0 ). Furthermore, the hydroxyl of Tyr236 (Phe15 in Tah1; Supplementary Figure 1 and Figure 2) contacts the main-chain carbonyl of Glu-3, an interaction not possible with Phe15 of Tah1, while the sidechain carbonyl group of Glu271 (Lys50 in Tah1) contacts the main-chain amide of Glu-3. The only interactions to side-chains (excluding the two carboxylate clamp) are between the guanidinium group of Arg305 and the side-chain amide of Asn308 (Lys 79 in Tah1) to the carboxylate group of Glu-3. Additionally, Val-1 is involved in a hydrophobic interaction with a pocket formed by Asn233, Tyr236 (Phe15 in Tah1), Asn264 and Ala267 (Met46 in Tah1), whereas the side-chain of Met-4 binds between Tyr236 (Phe15 in Tah1) and Glu271 (Lys50 in Tah1). Two further Van der Waals contacts involve the side chain of Glu-3 and Glu-0 with Phe270 (Ile49 in Tah1) and Thr260 (Val39 in Tah1), respectively. It should be noted that the Glu-2 side-chain is not involved in interactions. In summary, our alignments suggest that there are significant residue differences between the TPR2a-and the Tah1 TPR-domain that point to differences in the precise way they would bind the MEEVD peptide (Supplementary Figure 1 and Figure 2).

Ligand discrimination by Tah1 is similar to that of the TPR2a domain of Hop

For Hop the residue at position -4 of Hsp70 (I -4 EEVD 0 or VEEVD for SsaI) and Hsp90 (MEEVD) primarily determines the specificity for the TPR1-and TPR2a-domains, respectively [29]. To determine whether Tah1 ligand discrimination was similar to that of TPR2a we investigated the ability of Tah1 to bind peptides representing the C-terminal ends of Hsp90 and Ssa1. The results in Figure 3 show that the binding affinity for the C-terminal peptides of yeast Hsp90 increases with increasing peptide length (MEEVD, K d = 35.4 ± 4.4 µM; TEMEEVD, K d = 0.95 ± 0.04 µM; PADTEMEEVD, K d = 0.73 ± 0.04 µM; Figure 3a-c) and that the decapeptide most likely represents the intact binding site for Tah1 (PADTEMEEVD, K d = 0.73 ± 0.04 µM and full-length yeast Hsp90, K d = 0.78 ± 0.06 µM; Figure 3c and2a). Indeed, the binding of Tah1 to the human Hsp90 -and Hsp90decapeptide was also similar to that of the corresponding full-length proteins (Hsp90 decapeptide, DDTSRMEEVD, K d = 0.9 ± 0.06 µM; Hsp90 decapeptide, EDASRMEEVD,

The methione residue of the conserved MEEVD-motif confers tight Tah1 binding

The results so far show that Tah1 preferentially binds Hsp90 over Ssa1. Consequently, we next investigated the mechanism behind this selectivity. A comparison of the decapeptide sequences from a variety of cytoplasmic Hsp90s suggests that there is very little sequence conservation upstream of the conserved MEEVD-motif, except at the -6 position (91% conservation) and -5 position (68% conservation) [29]. This suggests that position -6 (which is mainly serine and rarely a threonine residue) may be critical for the specificity in the interaction with Tah1. Using ITC the binding of the PADAAMEEVD decapeptide (changes to wild type sequence are shown in italic; K d = 0.75 ± 0.04 µM; Figure 5a) was found to be similar to the decapeptide with wild type sequence (PADTEMEEVD, K d = 0.73 ± 0.04 µM; Figure 3c). Consequently, tight binding of Tah1 to Hsp90 is determined within the conserved MEEVD-motif, and upstream amino acids appear to be involved in mainly main-chain interactions. However, since the EEVD-motif is common to both Ssa1 and Hsp90 it follows that the conserved methionine of the MEEVD-motif is responsible for affecting tight binding to Tah1. Our results show that a decapeptide containing an alanine (PADTEAEEVD) or valine (PADTEVEEVD) in place of the conserved methionine in the MEEVD-motif significantly reduced the affinity for Tah1 (K d = 3.3 ± 0.2 and 9.8 ± 0.7 µM, respectively; Figure 5b,c) relative to wild type decapeptide (K d = 0.73 ± 0.04 µM; Figure 3c). Therefore, the results suggest that ligand discrimination for the MEEVD-peptide by Tah1 and the TPR2a domain of Hop [21] is similar in that both favour a methionine at position -4 of the MEEVD motif of Hsp90.

Tah1 positively discriminates against the valine at position -4 in the AEGPTVEEVDmotif of Ssa1

The results so far suggest that the first valine in the VEEVD-motif of Ssa1 prevents tight association with Tah1. Thus introducing an alanine or methionine residue at position -4 of the Ssa1 decapeptide should increase the binding affinity relative to a decapeptide containing a valine at this position. Our results show that this is indeed the case, where the decapeptides AEGPTAEEVD and AEGPTMEEVD, were found too bind with increasing affinity (K d = 14.8 ± 0.7 and K d = 4.2 ± 0.5 µM, respectively; Figure 5d,e) relative to the wild type sequence decapeptide (K d = 28.9 ± 3.1 µM; Figure 4c).

The M705V mutation of Hsp90 disrupts Tah1 binding

Having established with decapeptides that the methionine in the conserved MEEVD-motif of Hsp90 confers specificity for Tah1 binding we investigated whether this was also true for the intact yeast Hsp90. We therefore made the yeast Hsp90 M705V mutation to see whether it would bind Tah1 significantly less tightly. Firstly we tested the M705V mutation with CHIP, known to bind both Hsp90 and Hsp70, and then with the C-terminal domain of Sti1 (cSti1), which favours Hsp90 binding. As expected, the M705V mutation bound CHIP with an more or less equal affinity (K d = 6.7 ± 0.62 µM) relative to wild type Hsp90 (K d = 4.2 ± 0.63 µM; Figure 6a,b). Previous estimate for the CHIP and human Hsp90 interaction by ITC (K d =4.9 µM) is consistent with the value obtained here with yeast protein [24]. For cSti1 the M705V mutation bound less tightly (K d = 0.20 ± 0.03 µM) than the wild type protein (K d = 0.04 ± 0.01 µM; Figure 6c,d). It is noted that previous estimates with full-length Sti1 appear to be slightly lower in affinity (K d = 0.24 ± 0.07 and 0.33 ± 0.03 µM; [35,47]) against the wild type Hsp90, indicating that TPR1 may have a negative affect on the binding of the TPR2a-domain. However, having established that the M705V mutation affected the binding of both CHIP and the cSti1 in a predictable manner we next tested the binding of Tah1 and, as expected, we found that the M705V mutation inhibited the binding of Tah1 (K d = 13 ± 1.7 µM) relative to wild type protein (K d = 0.78 ± 0.06 µM; Figure 1a and6e).

Tah1 weakly stimulates the ATPase activity of Hsp90

TPR-domain containing proteins have been implicated in the regulation of the ATPase activity of Hsp90. The first report of such regulation was for Sti1, which was shown to be a potent inhibitor of the ATPase activity of Hsp90 [35]. In contrast, the TPR-domain containing co-chaperone, Cpr6, has been shown to have a weak stimulatory affect on the ATPase activity of Hsp90 [45]. Using a ten-fold excess of Tah1 we were able to show a very weak stimulation of the ATPase activity of Hsp90 (Figure 7a).

Tah1 appears to be primerally monomeric

TPR-domain containing proteins have been shown to be both monomeric (immunophillins such as, FKBP51 and Cpr6) and dimeric (Hop/Sti1 and CHIP) in nature [24,35,48]. We were interested in determining the oligomeric state of Tah1. We employed gel-filtration chromatography to determine the relative molecular weight of Tah1. Tah1 eluted, as a single peak, with a Mr (relative molecular weight) of 18,500 (Figure 7b). As Tah1 is basically a TPR-domain it is reasonable to expect this protein to be slightly elongated, rather than globular, in common with other TPR-domain motifs. Consequently, on gel-filtration it would appear slightly larger than 12.8 Kd (cleaved Tah1). Our gel-filtration results therefore suggest that Tah1 is most likely monomeric. To support this we conducted an analytical sedimentation equilibrium analysis on Tah1. Good results with high quality fits were obtained from both 7 and 0.5 mg ml -1 samples, and these are shown in Figure 7c and7d. The Tah 1 sample clearly cannot be simply monomeric, as the average molecular weight (See Table 2) by two independent methods confirms this. The excellent fit obtained with the INVEQ approach is essentially conclusive evidence that a dimeric species is present (estimated to be around 19%), but probably not higher oligomers, at least in significant quantity. The single experiment at 7 mg ml -1 is however not by itself capable of distinguishing between the presence of a dimer in reversible equilibrium with monomer, and an irreversible dimer being present. To make this distinction we must compare the two, in this case widely separated, solute concentrations. The predictions for the effect of dilution are that if a reversible dimer is present the K d value estimated will be the same, but the average molecular weight will decrease markedly. However, if an irreversible dimer is present the K d value estimated will increase markedly, but the average molecular weight will remain the same. Inspection of Table 2 shows immediately that it is the second hypothesis that is supported. The results therefore show that Tah1 is primerall y monomeric under the conditions used, but that some dimeric species is also present. The fact that the monomeric form is not in equilibrium with the dimeric species suggests that the dimer may not be a biologically native species, but rather aggregated molecules of Tah1. TPR-domains are known to self-associate [20] and we have in this study observed irreversible aggregation and precipitation of Tah1 on prolonged storage and during handling of the protein. Even though our samples were spun to remove such aggregates small soluble species might not be removed by centrifugation. We therefore conclude that Tah1 is most probably monomeric as a biologically active species and that the dimeric species is most probably irreversibly aggregated dimer.

DISCUSSION

This study has shown that the TPR-domain of Tah1 is similar to that of the TPR2b-domain of Hop. Tah1 was shown to selectively favour the binding of methionine over a valine at position -4, and therefore selectively binds Hsp90 over Ssa1, in common with the TPR2adomain of Hop. However, although tight binding of ligand by Tah1 and the TPR2a domain of Hop is dependant on a methionine at this position the overall interactions that these domains make with the MEEVD-motif are actually different since the sequence conservation between the two domains is significantly diverged. This is even more remarkable as many of the upstream contacts from the MEEVD-motif and contacts made by the TPR2a-domain with MEEVD appear to be man chain contacts to their respective peptides. The orientation of peptide binding in different TPR-domains has been shown to vary. For example the peptide in the TPR2a-MEEVD complex is bound in an antiparallel orientation to -helix 1, 3, 5 and 7 of the domain. However, in the Cyp40-and FKBP51-complexes the EEVD is bound in the opposite orientation [21][22][23]25]. Our amino acid sequence alignment showed that for residues involved in contacting the MEEVD-motif by the TPR2a-domain seven residues are identical and eight residues are different at equivalent positions in the Tah1-domain. Consequently, the orientation in which the MEEVD-peptide binds to Tah1 cannot be assumed to be the same as seen in the TPR2a-MEEVD complex, and must await structural determination to be resolved.

A high affinity-binding motif for the TPR2b domain of Hop has not been identified [21,29], but surprisingly Tah1 was shown to be most similar to The TPR2b domain of Hop. This suggests that differences between these two TPR-domains direct their selectivity towards different binding sites. It is interesting to note that the TPR2b-domain has been shown to be critical for the interaction of the TPR2a-domain of yeast Sti1 with Hsp90 [49].

It has also been reported that binding of TPR-domain containing co-chaperones to Hsp70 and Hsp90 can influence the ATPase activity of the chaperone [32][33][34][35][36]. Under the assay conditions that we applied we were able to detect a weak stimulation of the ATPase activity of Hsp90 with a ten-fold excess of Tah1. While this activation is very weak the ATPase activity of Hsp90 is critical to its proper functioning [46]. However, how TPR domain proteins bind the extreme C-terminus of Hsp90 and influence its ATPase activity is still not understood and may have to wait for structural studies to shed light on the precise mechanism.

centrifugation carried out by, and useful discussion with, Professor Arthur J. Rowe, NCMH Business Centre School of Biosciences, University of Nottingham. We also thank the KC Wong Foundation and the China Scholarship Council (CZ). ITC by injecting into Tah1 (a), the pentapeptide VEEVD; (b), the heptapeptide PTVEEVD and (c), the decapeptide AEGEPTVEED representing the extreme C-terminus of Ssa1. The results show that the Tah1 interaction with Ssa1 peptides, remains weak, and does not increase in affinity with increasing peptide length, in contrast to equivalent peptides based on Hsp90. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2008 as manuscript BJ20080105 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080105 S t a g e 2 ( a ) P O S T -P R I N T Relative molecular weight determination by Gel-filtration 0.5 ml aliquots of PreScission cleaved Tah1 (1.4 mg ml -1 ) were loaded onto a Superdex 75 HR 16/60 column equilibrated in 20 mM Tris-HCl pH 7.4, 1 mM EDTA and 250 mM NaCl.

  K d = 1.0 ± 0.19 µM; full length Hsp90 , K d = 0.33 ± 0.03 µM and full length Hsp90 , K d = Biochemical Journal Immediate Publication. Published on 15 Apr 2008 as manuscript BJ20080105 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080105 S t a g e 2 ( a ) P O S T -P R I N T 0.34 ± 0.07 µM; Figure 1c, d and 3d, e). In contrast to the Hsp90 decapeptides, peptides representing the C-terminal end of Ssa1 showed a significantly weaker affinity for Tah1 (VEEVD, K d = 48.8 ± 5.5 µM; PTVEEVD, K d = 10.6 ± 1.4 µM and AEGPTVEEVD, K d = 28.9 ± 3.1 µM; Figure 4a-c).

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2008 as manuscript BJ20080105 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080105 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2008 as manuscript BJ20080105 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080105 S t a g e 2 ( a ) P O S T -P R I N T

FIGURE LEGENDS Figure 1

 LEGENDS1 FIGURE LEGENDS

Figure 2

 2 Figure 2 Pymol diagram showing the interaction of the MEEVD peptide with the TPR2a domain of Hop.

Figure 3

 3 Figure 3 Isothermal titration calorimetry of Tah1 with Hsp90 peptides carrying the MEEVDmotif

Figure 4

 4 Figure 4 Isothermal titration calorimetry of Tah1 and Ssa1 peptides containing the VEEVDmotif.

Figure 5

 5 Figure 5 Isothermal titration calorimetry with Tah1 and peptides with altered Hsp90 and Ssa1 sequence

Figure 6

 6 Figure 6 Isothermal titration calorimetry of TPR-domain containing proteins and the Hsp90 M705V mutation

Figure 7

 7 Figure 7 Tah1 activation of Hsp90 and determination of the relative molecular weight of Tah1

Table 1 . Summary of the binding affinities of Tah1, Chip and cSti1.

 1

	Biochemical Journal Immediate Publication. Published on 15 Apr 2008 as manuscript BJ20080105
	Tah1 interactions	K d (µ M)	Stoichiometry
	with		(N)
	Yeast Hsp90	0.78±0.06	1.1
	C-terminal domain of	0.32±0.03	0.93
	Hsp90
	Hsp90	0.33±0.03	1.1
	Hsp90 Ssa1 YeastHsp90 peptides MEEVD TEMEEVD PADTEMEEVD PADAAMEEVD PADTEAEEVD PADTEVEEVD Human Hsp90 peptides DDTSRMEEVD EDASRMEEVD Ssa1 peptides VEEVD PTVEEVD AEGPTVEEVD AEGPTAEEVD AEGPTMEEVD Hsp90 and M705V	0.34±0.07 16±2.5 35.4±4.4 0.95±0.04 0.73±0.04 0.75±0.04 3.3±0.2 9.8±0.7 0.9±0.06 1.0±0.19 48.8±5.5 10.6±1.4 28.9±3.1 14.8±0.7 4.2±0.5	0.84 1.0 (fixed) 1.1 1.2 1.0 0.81 0.89 1.1 1.1 0.81 1.0 (fixed) 1.0 1.2 1.1 0.98	THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080105
	interactions
	CHIP-Hsp90	4.2±0.63	1.2
	CHIP-Hsp90 M705V 6.7±0.62	1.1
	cSti1-Hsp90	0.04±0.01	0.88
	cSti1-Hsp90 M705V	0.2±0.03	0.98
	Tah1-Hsp90 M705V	13±1.7	0.83

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

Table 2 . Summary of values for parameters estimated via sedimentation equilibrium analysis for Tah1 at two cell loading concentrations:

 2

	Protein	K d by	M(z) by	M(z) by	K d' by
	Concentration	INVEQ	Mfit	NONLIN'	NONLIN'
	(mg ml -1 )	(mM)		(kDa)	(mM)
	7.0	3.9	15.968	16.062	1.80
	0.5	0.51	16.106	15.845	0.15

Biochemical Journal Immediate Publication. Published on 15 Apr 2008 as manuscript BJ20080105 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080105 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.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 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

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080105 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. © 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. © The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We gratefully acknowledge support from the Wellcome Trust (LHP). We also thank Mrs. Jacky Metcalfe (ICR) for the peptide synthesis. We also acknowledge the analytical


Xavier Robellet

Michel Flipphi

Sylvine Pégot

Andrew P Maccabe

Christian Vélot
email: christian.velot@igmors.u-psud.fr

AcpA, a member of the GPR1/FUN34/YaaH membrane protein family, is essential for acetate permease activity in the hyphal fungus Aspergillus nidulans

Keywords: Monocarboxylate transport, acetate, Aspergillus nidulans, GPR1/FUN34/YaaH protein family, gene expression

In a previous study, alcS, a gene of the Aspergillus nidulans alc cluster, was shown to encode a protein that belongs to the GPR1/FUN34/YaaH membrane protein family. BLAST screening of the A. nidulans genome data identified additional genes encoding hypothetical proteins that could belong to this family. In this study we report the functional characterization of one of them, AN5226. Its expression is induced by ethanol and ethylacetate (two inducers of the alc genes) and is mediated by the specific transcriptional activator of genes of the acetate-utilization pathway FacB. Growth of a null mutant (∆AN5226) is notably affected when acetate is used as sole carbon source at low concentration and in a high pH medium, i.e. when protonated acetate, the form that can enter the cell by passive diffusion, is present in low amounts. Consistently, expression of AN5226 is also induced by acetate, but only when the latter is present at low concentrations. 14 C-Acetate uptake experiments using germinating conidia demonstrate an essential role for AN5226 in mediated acetate transport. To our knowledge this report is the first to provide evidence for the identification of an acetate transporter in filamentous fungi. We have designated AN5226 as acpA (for acetate permease).

INTRODUCTION

Like other filamentous fungi, Aspergillus nidulans is able to grow on two-carbon compounds, such as acetate and ethanol, as sole carbon sources. The ethanol utilization pathway of A. nidulans (termed the alc system) requires two structural genes, alcA and aldA.

These encode the enzymes alcohol dehydrogenase and aldehyde dehydrogenase, respectively, which catalyse the conversion of ethanol into acetate via acetaldehyde. Induction of alcA and aldA expression is controlled by a regulatory gene, alcR, encoding the pathway-specific autoregulated transcriptional activator [1]. To enter mainstream metabolism, acetate is further converted into acetyl-CoA by acetyl-CoA synthetase, encoded by facA [START_REF] Sandeman | Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans[END_REF][START_REF] Armitt | Analysis of acetate nonutilizing (acu) mutants in Aspergillus nidulans[END_REF]. Acetyl-CoA is either catabolized by the Krebs tricarboxylic acid (TCA) cycle in the mitochondrion or assimilated by the glyoxylate cycle, an anaplerotic version of the TCA cycle that is absolutely required for the utilization of two-carbon compounds when they are present as sole carbon sources. The glyoxylate pathway, present in bacteria, plants and fungi, enables the synthesis of C4 dicarboxylic acids from acetyl-CoA units, bypassing the two decarboxylation steps in the TCA cycle. The C4 metabolites so produced can be used for energy production, gluconeogenesis and other biosynthetic pathways. In A. nidulans, the two enzymatic activities specific to the glyoxylate cycle, isocitrate lyase and malate synthase, are encoded by acuD [START_REF] Ballance | Gene cloning in Aspergillus nidulans: isolation of the isocitrate lyase gene (acuD)[END_REF] and acuE [START_REF] Sandeman | Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans[END_REF][START_REF] Armitt | Analysis of acetate nonutilizing (acu) mutants in Aspergillus nidulans[END_REF], respectively. Unlike alcA, aldA and alcR, the acuD, acuE and facA genes are under the control of FacB [START_REF] Armitt | Analysis of acetate nonutilizing (acu) mutants in Aspergillus nidulans[END_REF][START_REF] Todd | The acetate regulatory gene facB of Aspergillus nidulans encodes a Zn(II)2Cys6 transcriptional activator[END_REF], the acetate utilization pathway-specific transcriptional activator.

alcR and alcA are clustered on the left arm of chromosome VII together with three other genes, alcO, alcM and alcS, that are subject to strict AlcR control [START_REF] Fillinger | A newly identified gene cluster in Aspergillus nidulans comprises five novel genes localized in the alc region that are controlled both by the specific transactivator AlcR and the general carbon-catabolite repressor CreA[END_REF]. However, their functions remain unknown. In a previous study [START_REF] Flipphi | Functional analysis of alcS, a gene of the alc cluster in Aspergillus nidulans[END_REF] we showed that alcS encodes a plasma membrane protein belonging to the GPR1/FUN34/YaaH family which includes among others, Gpr1p of Yarrowia lipolytica [START_REF] Kujau | Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase[END_REF] and Ady2p and Fun34p of Saccharomyces cerevisiae [START_REF] Rabitsch | A screen for genes required for meiosis and spore formation based on whole-genome expression[END_REF][START_REF] Stettler | An essential and specific subunit of RNA polymerase III (C) is encoded by gene RPC34 in Saccharomyces cerevisiae[END_REF]. While no clear biochemical function has yet been established for any protein in this family (Transporter Classification system: TC-9.B.33), phenotypic effects have been described in yeast species for gpr1 and ady2 mutants in relation to the utilization of acetate as the growth substrate. In S. cerevisiae, ADY2 expression (as well as that of FUN34) is elevated in liquid culture upon a shift from glucose medium to the non-fermentative growth substrate acetate. Deletion of ADY2 results in the loss of mediated acetate uptake, implying that this gene is necessary for the expression of an acetate permease [START_REF] Paiva | Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae[END_REF]. In Y. lipolytica, deletion of the GPR1 gene leads to a prolonged lag phase in growth on acetate [12]. Moreover, a recent study shows that certain mutations in ADY2 and FUN34 likewise induce acetic acid sensitivity in S. cerevisiae [13]. By contrast, A. nidulans alcS-deleted mutants do not display any obvious phenotype (on ethanol nor on acetate), suggesting that some other gene/genes might be able to compensate the absence of alcS under the conditions tested. A BLAST search of the A. nidulans genome [14] led to the identification of two novel genes that are induced by ethanol and ethylacetate (inducers of the alc system) and that could encode other members of the GPR1/FUN34/yaaH family, AN5226 and AN8390 [START_REF] Flipphi | Functional analysis of alcS, a gene of the alc cluster in Aspergillus nidulans[END_REF].

Here we report the functional characterization of AN5226, now designated AcpA (for acetate permease A). An acpA gene deletion mutant shows notably reduced growth when acetate is used as a sole carbon source at low concentration and in a high pH medium. 14 C-Acetate uptake experiments using germinating conidia lead us to identify AcpA as an essential component of mediated acetate transport.

EXPERIMENTAL

Strains, media, general growth conditions:

The A. nidulans strains used in this study are listed in Table 1. Genotypic annotations have been described previously [START_REF] Clutterbuck | Genetic Maps: Locus Maps of Complex Genomes[END_REF]. Media composition, supplements and basic growth conditions were as described by Cove [16].

Nitrogen sources were generally added to a final concentration of 10 mM nitrogen, i.e. diammonium tartrate and urea were each used at 5 mM. Carbon sources were added at either 0.1%, 0.5% or 1% (w/v or v/v) as specified.

General molecular techniques, classical genetics, transformation: Standard molecular

techniques were as described in Sambrook and Russell [17]. Aspergillus genomic DNA utilized for PCR reactions and in Southern blot analyses was isolated according to Specht et al. [18]. PCR products were purified using the High Pure PCR Product Purification Kit (Roche). Auxotrophic markers were exchanged and mutant alleles were combined through meiotic recombination employing conventional genetic techniques [START_REF] Clutterbuck | Handbook of genetics. Bacteria, Bacteriophages, and Fungi[END_REF]. Protoplast generation and transformation of A. nidulans with PCR-amplified DNA were performed essentially as described by Tilburn et al. [20].

Structure of the acpA gene:

Features of the acpA gene structure (intron positions, transcription start-and end-points) were revealed upon sequencing of cDNA clones obtained upon rapid amplification of cDNA ends (RACE) with a sample of total RNA. The RACE of acpA was performed with RNA isolated (according to the method described below) from a ∆alcS strain (CV171) induced with ethylacetate, utilizing a number of gene-specific oligonucleotide primers (Table 2). The GeneRacer kit from Invitrogen was used for the 5' RACE and the 5'/3' RACE kit 2 nd generation from Roche was used for the 3' RACE. 2). The oligonucleotides 5226-5'-pto3'-sht and 5226-3'-pto5'-sht have 28 and 30 base-long homologies with the 3' and 5' non-coding regions of the ketopantoate hydroxymethyl transferase (panB) gene [START_REF] Kurtov | The Aspergillus panB gene encodes ketopantoate hydroxymethyltransferase, required for biosynthesis of pantothenate and Coenzyme A[END_REF], respectively. Genomic DNA from strain BF107 was used as the template for PCR amplifications of the 5' and 3' acpA flanking regions. The panB gene was amplified using the oligonucleotide couple pto3'-5226-5'sht/pto5'-5226-3'sht (Table 2).

These oligonucleotides have 28-29 base-long homology with the 5' and 3' non-coding regions of the acpA gene. The plasmid pJVF089 carrying a functional A. nidulans panB gene [START_REF] Forment | Identification mstE gene encoding a glucose-inducible, low affinity glucose transporter in Aspergillus nidulans[END_REF] was used as the template for this PCR. After gel purification, equimolar amounts of the three fragments obtained were fused by means of a PCR without oligonucleotide primers under the following conditions: 5 min at 94°C; 15 cycles of (45 s at 94°C, 2 min at 58°C and 8 min at 68°C) and 15 min post-polymerisation with the Expand Long Template PCR System (Roche).

Subsequently, the oligonucleotide couple 5226-5'anti/5226-3'sens were added and the same cycling program was executed except that the number of cycles was increased to 25. Finally, a PCR was performed with nested oligonucleotides, Nes5226-3'sens and Nes5226-5'anti (Table 2), to generate the acpA::panB disruption cassette (~6.5 kb).

Generation of the null ∆acpA and the double ∆acpA ∆alcS mutants:

To generate the acpA knock-out mutant, 3 µg of the 6.5-kb acpA disruption cassette (see above) was used to transform the D-pantothenic acid-requiring panB100 strain CV164 and the ∆alcS panB100 strain CV171 (Table 1). Disruption of the acpA gene in a number of pantothenate prototrophic transformants was checked by Southern analysis. The resulting null ∆acpA and the double ∆acpA ∆alcS mutant strains used in this study were termed CV260 and CV253 (Table 1).

CV164 and CV171 were also transformed with the 2.5-kb panB fragment alone to generate the control strains CV312 and CV313 (i.e., pantothenate prototrophic transformants carrying a wild-type acpA gene -see Table 1).

Transcript analysis:

Mycelia for transcript analysis were grown for 24 h in appropriately supplemented minimal medium with lactose (3% w/v) as the carbon source and urea as the nitrogen source [START_REF] Flipphi | Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulans[END_REF]. Media were buffered with 100 mM sodium phosphate pH 6.8 added to cooled autoclaved medium from filter-sterilized stocks. Induction was achieved by the addition of the inducers to the concentrations indicated and cultures were harvested after 2.5 h of further incubation. Total RNA was isolated from mycelia as described previously [START_REF] Flipphi | Onset of carbon catabolite repression in Aspergillus nidulans. Parallel involvement of hexokinase and glucokinase in sugar signaling[END_REF].

Northern analysis was carried out with 10 µg samples of glyoxal-treated total RNA essentially as dscribed by Flipphi et al. [START_REF] Flipphi | Onset of carbon catabolite repression in Aspergillus nidulans. Parallel involvement of hexokinase and glucokinase in sugar signaling[END_REF]. DNA probes were labelled with 32 P dCTP using the Megaprime TM DNA Labelling System (Amersham Biosciences). Hybridisation signals were revealed using a PhosphorImager (Molecular Dynamics). The γ-actin (acnA) gene served as a reference as it is considered to be expressed equally well in all strains and under all conditions tested.

Acetate uptake measurements:

The isolation of germinating conidia and uptake measurements were performed essentially as previously described by MacCabe et al. [START_REF] Maccabe | Glucose uptake in germinating Aspergillus nidulans conidia: involvement of the creA and sorA genes[END_REF].

Acetate uptake rates were measured by assaying the incorporation of [1-14 C] acetic acid (sodium salt, with a specific activity of 2.11 GBq/mmol; GE Healthcare) into conidia at various external acetate concentrations. Uptake was quenched by rapid addition of 1.5 ml icecold unlabelled 1 M acetate in minimal medium at pH 6.8. The nitrocellulose filters used to collect the germinating conidia were rinsed with 1.5 ml of the same acetate solution immediately prior to sample filtration. Acetate uptake rates were determined by linear regression of plots of the amounts of radiolabelled acetate taken up by a fixed number of 4-h old glucose germlings after short periods of incubation (i.e. 5, 30, 60, 90, 120 and 180 seconds) at 37°C in a fixed volume under continuous agitation. All data were analysed using SigmaPlot Version 8.02 (SSP Inc.)

RESULTS

Structure of the acpA gene and its putative translation product

Using RACE, the exact position of the acpA transcripts was mapped by analyzing the sequences of a number of acpA-specific cDNA clones generated from the same RNA sample obtained from ethylacetate-induced mycelium (see Experimental). The acpA open reading frame (ORF) and its flanking regions is shown in Fig. 1A. The complete DNA sequence determined is available under the GenBank accession number EF428327. Sequencing of the acpA-cDNA identified two intron sequences disrupting the coding region of the gene. The coding sequence formed upon their removal would encode a putative product of 298 amino acids. Consistent with the two acpA RNA products observed on northern blots [START_REF] Flipphi | Functional analysis of alcS, a gene of the alc cluster in Aspergillus nidulans[END_REF], 5'-RACE experiments identified two 5' untranslated regions (5' UTR) of 398 and 247 bases in length.

However, the two transcriptional initiation sites are 469 bases apart. This discrepancy is due to the absence of a 318 base sequence in the 5' UTR of the longer transcript running from position -339 to position -22 (relative to the presumed start codon) (Fig. 1A and1B). This upstream intron sequence exhibits an unusual structure, in particular towards its 3' end, where it is heavily enriched in adenine residues organized in consecutive polyA stretches located between a 216 base ORF found within the intron and the presumed intron lariat sequence (GCUGAC). The transcription initiation site of the shorter RNA product is located within this 5' UTR intron (Fig. 1A and1B) hence the 5' UTR sequence is almost unique for each of the two transcripts, with the exception of the 21 bases immediately preceding the presumed start codon. To confirm the data obtained from these 5'-RACE experiments, we probed a northern blot made with total RNA isolated from ethylacetate-induced mycelia using probes specific for each of the acpA mRNA products (Fig. 1B). Whilst two transcripts are clearly produced (Fig. 1C), the absence of an upstream AUG in the 5' UTR of the longer mRNA make it likely that both transcripts give rise to the same translation product. The AcpA protein is a member of the GPR1/FUN34/YaaH membrane protein family (TC-9.B.33) [START_REF] Flipphi | Functional analysis of alcS, a gene of the alc cluster in Aspergillus nidulans[END_REF]. When the deduced amino acid sequence of AcpA is subject to analysis using algorithms designed to address membrane protein topology it is predicted to have six unambiguous transmembrane domains flanked by two additional putative domains. The six family-characteristic membrane spanning segments are indicated in Fig. 1A. The two putative membrane spanning segments (not depicted in the Figure) comprise residues 13 to 33 and 268 to 288.

Deletion of acpA affects growth on acetate as sole carbon source at low concentration and in a high pH medium

Using a gene replacement strategy, a complete loss-of-function ∆acpA single mutant was constructed as well as a ∆acpA ∆alcS double deletion mutant (see Experimental). The ability of these mutant strains to grow on different carbon sources was examined by plate testing. While no growth defects were observed on glucose, glycerol or ethanol, the ability of these mutants to utilize acetate appeared to be affected. Acetate is generally used at 0.5 % (w/v of the sodium salt) and Aspergillus media are normally set at pH 6.8, well above the pKa of acetic acid (4.75 at 25°C). [pKa is the pH value at which the dynamic pH-dependent equilibrium between the protonated (HA) and dissociated (A -) forms of acetate are present in equimolar concentration. At pH values below the pKa this equilibrium is shifted towards the protonated uncharged form whereas higher pH values result in predominance of the anion].

Plate growth was therefore tested not only at different acetate concentrations but also at different medium pH. As can be seen in Fig. 2, the ∆acpA mutants clearly manifest a growth defect at the low acetate concentration (0.1 %) which is more pronounced at high pH where the acid becomes increasingly limiting compared to the acetate anion. As could be expected, Interestingly, growth on acetate was further reduced in the double ∆acpA ∆alcS mutant compared to ∆acpA, whereas a ∆alcS mutant grew just as well as the wild-type strain (Fig. 2). A. nidulans is able to utilize agar as a very poor carbon source in the absence of any other growth substrate (i.e. under conditions of carbon starvation), leading to colonies of low hyphal density and sparse sporulation [START_REF] Armitt | Analysis of acetate nonutilizing (acu) mutants in Aspergillus nidulans[END_REF]. In this regard, starvation growth of a ∆alcS mutant on minimal medium agar plates lacking an added carbon source is compromised compared to that of a wild-type strain or a single ∆acpA null mutant (data not shown). This defect in the ∆alcS mutant could therefore explain the more severe phenotype observed for the double ∆acpA ∆alcS mutant on limiting acetate plates.

acpA is required for induction of the acetate-utilization pathway genes by limiting amounts of externally applied acetate

Considering the results presented above, we carried out northern blot experiments to test the ability of acetate to induce the expression of the genes required for acetate utilization, such as acuE, in the ∆acpA mutant when acetate is used at 0.1 % (i.e. 7.2 mM) in medium of pH 6.8 (Fig. 3A). At this pH the dissociated form (A -) represents more than 99% of the acetate molecules. While acuE is strongly induced under these conditions in a wild-type strain, no induction was observed in the null ∆acpA mutant. Deletion of acpA thus results in a phenotypic effect similar to that originally described by Kujau et al. [START_REF] Kujau | Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase[END_REF] for glyoxylate pathway regulator (gpr) mutations in Y. lipolytica, including among others gpr1 mutations. To confirm that the lack of acuE induction is due to the inability of acetate to enter the cell in this mutant, we also tested the ability of ethanol and ethylacetate (at the same low concentration) to induce acuE expression in the ∆acpA strain. Induction of the acetate-utilization pathway genes by ethanol and ethylacetate is due to the production of acetate resulting from the intracellular catabolism of these carbon sources [START_REF] Flipphi | Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulans[END_REF][START_REF] Flipphi | Characteristics of physiological inducers of the ethanol utilization (alc) pathway in Aspergillus nidulans[END_REF] and hence does not involve any acetate uptake. No differences in the ethanol and ethylacetate induction patterns were observed between the wild-type and the ∆acpA mutant strains (Fig. 3B). Conversely, ethanol induction of acuE is absent in alcR mutants (data not shown) as these cannot catabolize ethanol into acetate [1]. These data support the hypothesis that AcpA is involved in the uptake of the dissociated form of acetate.

Expression of the acpA gene is induced by low concentrations of acetate

In a previous study we reported that acpA (AN5226) itself is not induced by acetate but rather expressed at a basal level similar to that observed on lactose [START_REF] Flipphi | Functional analysis of alcS, a gene of the alc cluster in Aspergillus nidulans[END_REF]. However, in those experiments acetate was used at a concentration of 36 mM (0.5%) in non-pH stabilised medium. The present results (described above) indicate that acpA is required primarily when the protonated form of acetate (HA) is present at limiting concentrations that are insufficient for growth. We therefore analysed transcriptional expression of acpA in a wild-type strain grown at pH 6.8 in response to increasing concentrations of acetate (from 7.2 mM to 36 mM, i.e. from 0.1 to 0.5%) using acuE as a control. As shown in Fig. 4 acpA is clearly induced by low amounts of acetate, its level of expression apparently being inversely correlated to the acetate concentration; up to 28 mM, acuE induction appears to follow the opposite trend. This acpA transcription profile can account for the growth phenotypes shown in Fig. 2.

The dramatic drop in the acuE transcript level observed between 28 and 36 mM of acetate in medium phosphate-buffered at pH 6.8, is most probably a consequence of acetate toxicity. Previously we have noticed that a similar drop in the γ-actin (acnA) transcript level occurs between 36 and 40 mM in medium set at pH 6.8 (but not phosphate buffered) and that this effect occurs at increasingly lower acetate concentrations with decreasing medium pH (data not shown).

Transcription pattern of acpA upon addition of various weak carboxylic acids

To further specify the induction spectrum of acpA we performed northern blot analysis on RNA isolated from wild-type mycelia supplied with 7.2 mM (final concentration) of various weak monocarboxylic acids: lactate, pyruvate, propionate, formate and glyoxylate. Of these, only addition of propionate results in significant induction of acpA (Fig. 5).

Interestingly, this induction is much weaker when propionate is supplied at 36 mM (data not shown), suggesting that the inducing ability of this weak acid is similar to that of acetate (i.e. it provokes transcriptional induction of acpA when present in limiting amounts). While growth was extremely poor on these monocarboxylic acids as sole carbon sources, no significant differences in growth were observed between the ∆acpA null mutants and the wild-type (data not shown). Moreover, testing propionate toxicity using a wide range of propionate concentrations on glycerol minimal medium plates (at pH 6.8) did not discriminate acpA-deleted strains from wild-types (data not shown). These data suggest that AcpA has a restricted substrate range and that acetate is its physiologically relevant substrate.

Transcriptional activation of acpA

Intriguingly, while the acpA response to glyoxylate was rather weak at low concentrations (Fig. 5) this intermediate of acetate catabolism provoked a powerful induction at 50 mM (data not shown) -the reverse of that observed for acetate and propionate. It has previously been shown that glyoxylate at 50 mM is also good inducer of the transcription of the acetyl-CoA synthethase gene facA, one of the structural genes under the control of the acetate pathway-specific activator FacB [START_REF] Flipphi | Characteristics of physiological inducers of the ethanol utilization (alc) pathway in Aspergillus nidulans[END_REF]. These observations prompted us to investigate whether the induction of acpA is dependent on the function of FacB. To address this we compared the expression of acpA between a wild-type strain and a facB mutant (facB101) after induction with ethanol and ethylacetate and used acuE as an expression control. The lack of acpA induction (Fig. 6) in the complete loss-of-function mutant facB101 [START_REF] Todd | Molecular characterization of mutants of the acetate regulatory gene facB of Aspergillus nidulans[END_REF] correlates with the presence of six putative binding sites for the binuclear zinc-cluster protein FacB [START_REF] Todd | FacB, the Aspergillus nidulans activator of acetate utilization genes, binds dissimilar DNA sequences[END_REF] in the promoter of the acpA gene: two sites located upstream of the distal transcriptional start and four more located upstream of the proximal start site (Fig. 1A). The induction pattern of acpA in a facA mutant (facA330) -the latter cannot convert acetate into acetyl-CoA -is similar to that observed in the wild-type strain, indicating that the loss of induction in the facB101 mutant is due to the absence of functional FacB and not a consequence of the loss of the ability to catabolize acetate. It should be noted that our analysis suggests that the basal level (i.e. non-induced) expression of both acpA transcripts does not require FacB.

acpA is essential for acetate permease activity

To assess the role of acpA in acetate transport the uptake of 14 C-labelled acetate was compared between wild-type and ∆acpA null mutant conidia germinating in the presence of either glucose or ethanol as sole carbon sources, culture conditions known to result in acpA expression (data not shown). As can be seen in Fig. 7 acetate uptake by ∆acpA conidia is very considerably reduced compared to that of wild-type conidia. This is especially true at the lower acetate concentrations where the differences in calculated uptake rates range from 30 fold at 1 mM acetate to >100 fold at acetate concentrations between 1 -100 µM. To characterise the kinetics of acetate uptake the incorporation of 14 C-acetate by wild-type conidia germinating in glucose at pH 6.8 was measured across a wide range of substrate concentrations (1 µM -7 mM). Rates of uptake were obtained by linear regression of plots of acetate incorporation versus time. Rates plotted against substrate concentration showed Michaelis-Menten saturation kinetics from which a Km of ~230 µM was calculated (Fig. 7 inset). Eadie-Hofstee and Hanes-Wolf representations of the data were monophasic hence indicating that no additional system of acetate incorporation contributed significantly to its uptake. These data are consistent with the growth phenotype of the ∆acpA mutants on limiting amounts of acetate being due to an impairment of the uptake of the acetate anion, and the likelihood that AcpA functions as an acetate permease in A. nidulans.

DISCUSSION

In this paper we identify acpA (Broad annotation: AN5226) to encode a novel element in the biochemical pathway for acetate utilization in the filamentous fungus Aspergillus nidulans. The acpA gene is located on chromosome V but it is not closely physically linked to any of the five other loci involved in acetate metabolism that also reside on this chromosome: facA (AN5626), acuD (AN5634), acuG (AN5604), acuH (AN5336) and acuN (AN5746) [START_REF] Armitt | Analysis of acetate nonutilizing (acu) mutants in Aspergillus nidulans[END_REF].

Analysis of the sequence of the deduced translation product (AcpA) is indicative of a membrane related function since it bears homology to the defining members of the GPR1/FUN34/YaaH family (TC-9.B.33; Pfam PF01184) of membrane proteins [12,[START_REF] Kim | Topology models for 37 Saccharomyces cerevisiae membrane proteins based on C-terminal reporter fusions and predictions[END_REF][START_REF] Daley | Global topology analysis of the Escherichia coli inner membrane proteome[END_REF], and its hydrophobicity profile indicates the presence of six transmembrane domains.

Unlike other activities involved in acetate utilization, AcpA is dispensable for growth on the acetate precursor ethanol. Whilst this is also largely the case when 1% acetate is used as the sole carbon source, at reduced concentrations the growth of AcpA loss-of-function mutants (ΔacpA) is seen to become progressively impaired and this effect is further exacerbated at elevated pH. Previous studies of acetate uptake kinetics in yeast have provided evidence for distinct means of uptake for each of the two forms of acetate (i.e. HA and A -).

Entry of the anion into the cell is mediated by a transport system whilst the undissociated acid is able to freely diffuse across the plasma membrane [32,[START_REF] Casal | Utilization of short-chain monocarboxylic acids by the yeast Torulaspora delbrueckii: specificity of the transport systems and their regulation[END_REF]. The very poor growth observed for A. nidulans ∆acpA mutants on acetate under conditions in which the availability of the protonated (HA) form is very reduced or negligible (i.e. low acetate concentration and pH values considerably higher than the pKa) could thus be indicative of a defect in the transport of the anionic form. This is coherent with the observation of normal growth of ∆acpA mutants on ethanol since acetate is generated intracellularly upon catabolism of ethanol.

In addition to the growth phenotype on acetate, deletion of acpA results in altered regulation of the malate synthase (acuE) gene. Whilst acuE inducibility upon growth of ∆acpA mutants on acetate precursor substrates is unchanged from that of wild-type, its induction by limiting amounts of externally supplied acetate (0.1% at medium pH 6.8) is absent. Furthermore, the acpA transcript level in wild-type strains increases as the external acetate concentration decreases, i.e. the gene's response coincides with the need for a means to take up the acetate anion. These results are all consistent with the loss of an acetate uptake function in the acpA deletion mutant. Indeed, direct measurement of acetate incorporation into germinating conidia confirmed the effect of the acpA deletion on acetate uptake. At low substrate (acetate) concentrations acetate uptake is almost completely lost in acpA-deleted conidia compared to that seen in wild-type.

It has previously been postulated [START_REF] Semighini | Multi-copy suppression of an Aspergillus nidulans mutant sensitive to camptothecin by a putative monocarboxylate transporter[END_REF] that a gene (GenBank accession AY236409) corresponding to the A. nidulans Broad locus AN2840 could encode a monocarboxylate transporter (MCT) based on its similarity to functional mammalian MCTs. However, in S. cerevisiae of the five proteins that show substantial similarity to human MCT transporters none is important for the transport of carboxylic acids across the plasma membrane, and most are instead found associated with intracellular structures [START_REF] André | The putative monocarboxylate permeases of the yeast Saccharomyces cerevisiae do not transport monocarboxylic acids across the plasma membrane[END_REF]. To our knowledge, AcpA is thus the first experimentally supported candidate for a MCT in filamentous fungi.

By reciprocal TBLASTN screening we searched for AcpA orthologues in publicly available fungal genome databases. In all cases except that of the Chytridiomycota at least one putative orthologue of acpA could be identified. Fig. 8 shows a phylogenetic tree of those proteins most similar to AcpA in 27 fungi. The proteins identified in the Aspergilli share 61% identity and 74% similarity. These data indicate conservation of AcpA-like proteins and by inference the means of direct cellular uptake of the acetate ion.

With regard to their structural organisation, all Aspergillus spp acpA orthologues appear to contain canonical splicing sites [START_REF] Kupfer | Introns and splicing elements of five diverse fungi[END_REF] in their 5' UTR sequences that encompass a region highly enriched in A residues (for A. nidulans see Fig. 1A nucleotide position -72 to -35). The apparent occurrence of an intron upstream of the coding region in all Aspergilli acpA orthologues suggests not only structural conservation within the 5' UTR sequences but also a specific function for the intron. A similar organization is known for the A. nidulans gene brlA which encodes a key regulator of asexual development. The molecular basis for the production of two distinct brlA transcripts and translation products has been studied [START_REF] Prade | The Aspergillus nidulans brlA regulatory locus consists of overlapping transcription units that are individually required for conidiophore development[END_REF][START_REF] Han | Translational repression of brlA expression prevents premature development in Aspergillus[END_REF].

In the case of acpA however, the absence of any ATG in the 5' UTR of the longer transcript would suggest that both mRNA species probably give rise to the same translation product. This raises the question of the roles of the two acpA transcripts. One possibility can be illustrated by the S. cerevisiae JEN1 gene [START_REF] Casal | The lactateproton symport of Saccharomyces cerevisiae is encoded by JEN1[END_REF]. For this gene, one of the several different transcription products that result from multiple initiation transcription sites was shown to modulate the degradation of all JEN1-specific mRNAs [START_REF] Andrade | Multiple transcripts regulate glucose-triggered mRNA decay of the lactate transporter JEN1 from Saccharomyces cerevisiae[END_REF]. Another gene with an intron in its 5'UTR is the arginase encoding gene agaA of A. nidulans [START_REF] Borsuk | Structure of the arginase coding gene and its transcript in Aspergillus nidulans[END_REF]. This intron leads to the generation of various mRNA isoforms that influence not only mRNA stability but also their activities as templates for translation by a riboswitch mechanism [START_REF] Borsuk | L-Arginine influences the structure and function of arginase mRNA in Aspergillus nidulans[END_REF]. However, unlike acpA, the different agaA mRNA species result from alternative splicing.

In S. cerevisiae two functional MCTs have been described: a high-affinity transporter shared by lactate, acetate, propionate and pyruvate [43], and a second transporter of lower affinity and more restricted substrate specificity since it only transports acetate, propionate and formate [32]. While the former was found to be Jen1p (TC-2.A.1.12.2) [START_REF] Casal | The lactateproton symport of Saccharomyces cerevisiae is encoded by JEN1[END_REF][START_REF] Andrade | Expression of the lactate permease gene JEN1 from the yeast Saccharomyces cerevisiae[END_REF] a membrane protein that is assigned to the Major Facilitator Superfamily (TC-2.A.1), the latter depends on the integrity of the yeast acpA orthologue, ADY2 [START_REF] Paiva | Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae[END_REF].

By screening the A. nidulans genome sequence [14] we identified two putative gene products similar to S. cerevisiae Jen1p encoded by loci AN6703 and AN6095. The deduced proteins share 39% and 30% identity with Jen1p, respectively. Northern blot analysis upon addition of various monocarboxylic acids or ethanol to lactose-grown wild-type mycelia, failed to reveal expression of AN6095 (unpublished data). By contrast, AN6703 appeared to be modestly expressed upon formate and propionate induction but no response could be observed upon addition of acetate to 7.2 mM or 36 mM (0.1% -0.5%) nor in the presence of ethanol (i.e. under conditions inducing for the acpA gene) (unpublished data). These observations would explain why conidia of the null ∆acpA mutant germinated on glucose or ethanol are barely able to take up acetate under our experimental conditions (medium pH of 6.8 or higher -see Fig. 7). Alternative explanations could be that AN6703, the A. nidulans protein most similar to the yeast transporter Jen1p, is either unable to transport acetate oras is the case for ethanol -it is not expressed on glucose.

Together with previous yeast studies [START_REF] Paiva | Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae[END_REF][12][13], our data demonstrate a link between members of the GPR1/FUN34/YaaH protein family (TC-9.B.33) and acetate utilisation, suggesting that other members of this family, including A. nidulans AlcS and orthologues in other filamentous fungi, might be involved in acetate adaptation, uptake and/or catabolism. The shift from lower case to upper case corresponds to the junction between sequences from acpA and panB. were realized with RNA isolated from a ∆alcS strain (CV171) induced with 50 mM ethylacetate (see Experimental). The blots were subsequently hybridised with the appropriate 32 P-labelled probes (see Fig. 1B).

Name

Figure 2:

The acpA gene is required for growth on acetate minimal medium plates.

Plates with appropriately supplemented minimal medium were prepared containing sodium nitrate (10 mM) as nitrogen source and sodium acetate at the indicated concentration as the carbon source. The media were buffered at the indicated pH with 100 mM sodium phosphate (final concentration). Inoculated plates were incubated at 37°C for 2 days. The relative availabilities of the protonated (HA) and anionic (A -) forms are given by the [HA]:[A -] ratio for each pH. (A) Plates were point inoculated with four strains which differ only in the indicated deletion: the single mutants ∆acpA (strain CV260) and ∆alcS (strain CV313), and the double mutant ∆acpA∆alcS (strain CV253); strain CV312 was used as the control (see Experimental details are as described in the legend to Figure 3. Gene expression was induced with acetate across a range of concentrations from 7.2 mM (0.1%) to 36 mM (0.5%). The wild-type strain used was CV312 (see Table 1). Experimental details are as described in the legend to Figure 3. The indicated compounds were added to mycelial lactose cultures to a final concentration of 7.2 mM. The wild-type strain used was CV312 (see Table 1). The tree was rooted using protein sequence RO3G_03025 of Rhizopus oryzae as an outgroup.

The bootstrap probabilities (calculated using 100 replications) are indicated on each node.

For those organisms where a database of deduced proteins was accessible, the corresponding protein annotation code is given (provided that the predicted protein is identical to that we deduced by manual analysis).

23

4

  Biochemical Journal Immediate Publication. Published on 27 Feb 2008 as manuscript BJ20080124 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080124 S t a g e 2 ( a ) P O S T -P R I N T Construction of the acpA::panB disruption cassette: A gene replacement strategy, based on the principle of double joint PCR [21], was designed to obtain acpA complete loss-of-function mutants. The 5' and 3' flanking sequences of acpA were amplified using the oligonucleotide couples 5226-5'anti/5226-5'-pto3'-sht and 5226-3'sens/5226-3'-pto5'-sht (Table

7

  Biochemical Journal Immediate Publication. Published on 27 Feb 2008 as manuscript BJ20080124 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080124 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.

  re-introduction of the acpA gene into these gene-deleted mutants fully restored wild-type growth characteristics (data not shown).

10 Biochemical

 10 Journal Immediate Publication. Published on 27 Feb 2008 as manuscript BJ20080124 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080124 S t a g e 2 ( a ) P O S T -P R I N T

  Augstein, A., Barth, K., Gentsch, M., Kohlwein, S. D. and Barth, G. (2003) Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica, Microbiology 149, 589-600 13 Gentsch, M., Kuschel, M., Schlegel, S. and Barth, G. (2007) Mutations at different sites in members of the Gpr1/Fun34/YaaH protein family cause hypersensitivity to acetic acid in Saccharomyces cerevisiae as well as in Yarrowia lipolytica, FEMS Yeast Res. 7, 380-390

Figure legends Figure 1 :

 legends1 Figure legends

Figure 4 :

 4 Figure 4: Induction of acpA is inversely correlated to the acetate concentration.

Figure 5 :

 5 Figure 5: acpA expression in the presence of various weak monocarboxylic acids.

Figure 6 :

 6 Figure 6: Involvement of the transcriptional activator of acetate catabolism FacB in acpA induction. Experimental details are as described in the legend to Figure 3 except that the final concentration of ethanol or ethylacetate was 50 mM. Strains ED027 and CV061 provided facB and facA mutant backgrounds, respectively. CV160 was the wild-type strain used.

Figure 7 :

 7 Figure 7: AcpA is required for acetate uptake at pH 6.8. Acetate uptake by WT (CV312) or ∆acpA (CV260) conidia germinating in liquid glucose minimal medium (see Experimental) was determined at different concentrations of acetate (1, 10, 100, 1000 and

Figure 8 :

 8 Figure 8: Neighbour-joining phylogenetic analysis of fungal AcpA orthologues. AcpA orthologue proteins were identified by TBLASTN screening of the genome species: Neosartorya fischeri, Aspergillus fumigatus, Aspergillus clavatus, Aspergillus oryzae, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Uncinocarpus reesii, Coccidioides immitis, Histoplasma capsulatum, Ascosphaera apis, Botryotinia fuckeliana, Botrytis cinerea, Sclerotinia sclerotiorum, Septoria nodorum, Chaetomium globosum, Podospora anserina, Neurospora crassa, Fusarium verticillioides, Fusarium graminearum, Trichoderma reesei, Saccharomyces cerevisiae, Yarrowia lipolytica, Ustilago maydis, Coprinus cinereus and Rhizopus oryzae. A sequence alignment was generated using the CLUSTALW multiple sequences alignment program [48] and the PHYLIP package [49].

Biochemical

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Table 1 . A. nidulans strains used in this study.

 1

	Strain Genotype

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 . Oligonucleotides used in this study.

 2

Table 1

 1

). 21 Biochemical Journal Immediate Publication. Published on 27 Feb 2008 as manuscript BJ20080124 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080124 S t a g e 2 ( a ) P O S T -P R I N T

Figure 3: Malate synthase gene (acuE) transcription in the presence of a limiting amount of acetate depends on the integrity of the acpA gene.

  Fungal biomass was generated in lactose/urea liquid media and gene expression was induced/repressed with the indicated compounds by addition to mycelial cultures to a final concentration of 7.2 mM.

	Media were buffered at pH 6.8 with 100 mM sodium phosphate. Total RNA was isolated,
	denatured, separated and transferred to a nylon membrane as described in Experimental.
	Northern blots were subsequently hybridised with 32 P-labelled probes corresponding to the
	indicated genes (see Experimental). γ-actin gene (acnA) served as an internal control.
	CV260 provided the ∆acpA mutant background and CV312 served as the wild-type strain
	(see Table 1). (A) For induction, acetate was added to the mycelial cultures to a final
	concentration of 7.2 mM (0.1%). The control, NI, received no inducer compound. (B) The
	same experiment was carried out except that ethanol or ethylacetate were added for induction
	instead of acetate.

Biochemical Journal Immediate Publication. Published on 27 Feb 2008 as manuscript BJ20080124 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080124 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

© 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

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 27 Feb 2008 as manuscript BJ20080124 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080124 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

This work was supported by the Centre National de la Recherche Scientifique (CNRS: contrat ATIP 2JE077). X.R. was a recipient of doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche of the French Government and is currently supported by The Human Earth Foundation. M.F. is the recipient of a postdoctoral contract Ramón y Cajal from the Spanish Ministerio de Educación y Ciencia. We thank Drs Nathalie Oestreicher, Béatrice Felenbok and Claudio Scazzocchio for fruitful discussions, and Dr. Barry Holland for correcting the English.