Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans

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Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans

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Joana Sá-Pessoa a,1, Sotiris Amillis b, Margarida Casal a,⇑, George Diallinas b,⇑

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a b

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, Braga 4710-057, Portugal Faculty of Biology, Department of Botany, University of Athens, Panepistimioupolis, Athens 15781, Greece

a r t i c l e

i n f o

Article history: Received 7 March 2014 Revised 6 February 2015 Accepted 10 February 2015 Available online xxxx Keywords: Filamentous fungi Carbon catabolism Acetate Germination Specificity

a b s t r a c t AcpA has been previously characterized as a high-affinity transporter essential for the uptake and use of acetate as sole carbon source in Aspergillus nidulans. Here, we follow the expression profile of AcpA and define its substrate specificity. AcpA-mediated acetate transport is detected from the onset of conidiospore germination, peaks at the time of germ tube emergence, and drops to low basal levels in germlings and young mycelia, where a second acetate transporter is also becoming apparent. AcpA activity also responds to acetate presence in the growth medium, but is not subject to either carbon or nitrogen catabolite repression. Short-chain monocarboxylates (benzoate, formate, butyrate and propionate) inhibit AcpA-mediated acetate transport with apparent inhibition constants (Ki) of 16.89 ± 2.12, 9.25 ± 1.01, 12.06 ± 3.29 and 1.44 ± 0.13 mM, respectively. AcpA is also shown not to be directly involved in ammonia export, as proposed for its Saccharomyces cerevisiae homologue Ady2p. In the second part of this work, we search for the unknown acetate transporter expressed in mycelia, and for other transporters that might contribute to acetate uptake. In silico analysis, genetic construction of relevant null mutants, and uptake assays, reveal that the closest AcpA homologue (AN1839), named AcpB, is the ‘missing’ secondary acetate transporter in mycelia. We also identify two major short-chain carboxylate (lactate, succinate, pyruvate and malate) transporters, named JenA (AN6095) and JenB (AN6703), which however are not involved in acetate uptake. This work establishes a framework for further exploiting acetate and carboxylate transport in filamentous ascomycetes. Ó 2015 Published by Elsevier Inc.

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1. Introduction Carbon source uptake and metabolism is directly reflected in fungal growth, development and virulence, and is thus crucial for the exploitation of fungi in biotechnological applications for the production of secondary metabolites, or for understanding the mechanisms underlying fungal susceptibility to antifungals (Hynes et al., 2011). The increasing number of species with their genome sequence publicly available allows a better knowledge to be obtained concerning the versatility and intrinsic complexity of carbon metabolism in fungi (Flipphi et al., 2009). Among fungi, the genus Aspergillus includes more than 20 species with their genomes sequenced (http://www.aspgd.org/). Aspergillus species can utilize a diversity of organic compounds as carbon sources including two-carbon compounds, such as acetate and ethanol, or other ⇑ Corresponding authors. E-mail addresses: [email protected] (M. Casal), [email protected] (G. Diallinas). 1 Present address: Centre for Infection and Immunity (CII), Queen’s University Belfast, Northern Ireland, UK.

short-chain monocarboxylates (Armitt et al., 1976). A prerequisite for the utilization of these nutrients is the existence of efficient transport systems, with the exception of ethanol, which enters cells by simple diffusion. Monocarboxylic acid transporters have been identified and well-studied in different yeasts (Casal et al., 2008; Abbott et al., 2009), but little is known about carboxylic acid transporters in filamentous fungi. An anion selective channel (AnBEST1) permeable to citrate and a range of other organic ions including propionate and benzoate was recently identified in Aspergillus nidulans (Roberts et al., 2011). AmcA, a member of the Monocarboxylate Transporter (MCT) family, is transcriptionally induced by acetate, lactate or pyruvate only in the absence of glucose and might play a critical role in monocarboxylate transport and drug efflux (Semighini et al., 2004). The AlcS protein from the Acetate Uptake Transporter (AceTr) family was found to be located at the plasma membrane and be highly induced by ethanol and repressed by glucose (Fillinger and Felenbok, 1996), although not involved in the transport of ethanol, acetate or acetaldehyde (Flipphi et al., 2006). Another member of this family, AcpA, is the only acetate transporter characterized to date (Robellet et al., 2008).

http://dx.doi.org/10.1016/j.fgb.2015.02.010 1087-1845/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: Sá-Pessoa, J., et al. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.02.010

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The AcpA transporter has been shown to be essential for growth of A. nidulans at low acetate concentrations and high medium pH values, conditions under which the protonated form of the acid is limited (Robellet et al., 2008). Comparison of acetate transport in gene-deleted strains and wild type A. nidulans confirmed its role as an acetate transporter (Robellet et al., 2008). Induction of transcription of this gene by limiting concentrations of acetate and propionate is under control of the acetate catabolic pathway activator FacB, although this is not the case at high concentrations (Robellet et al., 2008). Expression of acpA is also induced in the presence of several weak monocarboxylic acids such as glyoxylate, propionate, lactate, pyruvate and formate (Robellet et al., 2008). The orthologue of AcpA in the yeast Saccharomyces cerevisiae is Ady2p, which is responsible for the uptake of acetate, propionate and formate in symport with protons (Casal et al., 1996; Paiva et al., 2004; Pacheco et al., 2012). This protein is also proposed to be involved in ammonia export (Palková et al., 2002; Váchová et al., 2009). The evidence for that is based on the observation that null ady2 mutants have reduced ammonia production when cells are growing in colonies (Palková et al., 2002), which has also been correlated with ammonia release upon fluorescent-tagged Ady2p appearance at the plasma membrane (Váchová et al., 2009). No studies have addressed the substrate specificity profile of AcpA or its role in ammonium efflux in A. nidulans. The present work had two goals. The first was to define the substrate specificity profile of AcpA and its role in ammonium export. For doing that, we first needed to identify the development stage and physiological conditions that maximize AcpA-mediated transport in relation to a background of other acetate-specific, unknown, transporters. Our second goal was related to the identification of other transporters that might be involved in acetate transport. Our results establish that AcpA is the major acetate transporter during germination of A. nidulans and define its specificity profile toward other short-chain carboxylates. In addition we identify a secondary acetate transporter (AcpB) operating mostly in mycelia and two other short-chain carboxylic acid transporters, JenA and JenB, which however do not contribute to acetate uptake.

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2. Materials and methods

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2.1. Media, strains, growth conditions, and transformation Standard complete (CM) and mineral media (MM) for A. nidulans were based on those described by Cove (1966). Nitrogen sources were used at the final concentrations of 5 mM urea or 10 mM di-ammonium tartrate. Glucose or other carbon sources were used at the final concentrations of 1% (w/v) or in the concentrations specified in the text. CM was used for cell maintenance and conidiospore production, and MM for growth tests and uptake assays. The pH of all liquid media was adjusted to 6.8 before autoclaving. The media for growth tests were buffered at the indicated pH with 100 mM final concentration sodium phosphate. Cells were cultured at 37 °C unless otherwise stated. Cell viability was accessed by counting the amount of conidiospores per mL using a Neubauer counting-chamber slide and assessing the number of viable conidiospores after standard serial dilutions (10-fold dilution series in PBS with 0.1% Tween 80) and plating in duplicate on CM. Viability was assessed by percentage of conidiospores counted in duplicate in the Neubauer counting-chamber slide that originated single colonies in CM. The strains used to characterize AcpA are described in Table 1. Gene replacement null mutant strains were constructed by transformation of pyrG89 riboB2 nkuAD strains (uracil/uridine, or riboflavin auxotrophies and DNA helicase null mutant, respectively; Nayak et al., 2006), using linear DNA cassettes containing flanking sequences of the relevant ORFs, separated by the Aspergillus fumigatus selection marker AFpyrG

Table 1 Aspergillus nidulans strains used in this work. Strain

Genotype

Reference

WT

pabaA1 pantoB100 riboB2 Tr. panB

acpAD

pabaA1 pantoB100 riboB2 Tr. acpAD::panB

NNO2F7 (WT) acpAD

nkuAD::argB pyrG89 pyroA4 riboB2

Robellet et al. (2008) Robellet et al. (2008) Nayak et al. (2006) TNO2A7  acpAD

acpBD acpCD jenAD jenBD acpAD acpBD acpAD acpCD acpAD jenBD acpAD jenAD acpBD acpCD jenBD jenAD acpAD acpBD acpCD acpAD jenAD jenBD

acpAD::panB nkuAD::argB pantoB100 pyrG89 pyroA4 riboB2 AN1839D::AFpyrG nkuAD::argB pyrG89 pyroA4 riboB2 AN7317D::AFryboB nkuAD::argB pyrG89 pyroA4 riboB2 AN6095D::AFpyrG nkuAD::argB pyrG89 pyroA4 riboB2 AN6703D::AFryboB nkuAD::argB pyrG89 pyroA4 riboB2 AN1839D::AFpyrG acpAD::panB nkuAD::argB pantoB100 pyrG89 pyroA4 riboB2 AN7317D::AFriboB acpAD::panB nkuAD::argB pantoB100 pyrG89 pyroA4 riboB2 AN6703D::AFriboB acpAD::panB nkuAD::argB pantoB100 pyrG89 pyroA4 riboB2 AN6095D::AFriboB acpAD::panB nkuAD::argB pantoB100 pyrG89 pyroA4 riboB2 AN1839D::AFpyrG AN7317D::AFriboB nkuAD::argB pyrG89 pyroA4 riboB2 AN6703D::AFriboB AN6095D::AFpyrG nkuAD::argB pyrG89 pyroA4 riboB2 acpAD::panB AN1839D::AFpyrG AN7317D::AFriboB nkuAD::argB pantoB100 pyrG89 pyroA4 riboB2 acpAD::panB AN6703D::AFriboB AN6095D::AFpyrG nkuAD::argB pantoB100 pyrG89 pyroA4 riboB2

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(Afu2g0836) or AFriboB (Afu1g13300) and cloned into the pGEM T-Easy vector. Double and triple mutants were generated by transformation of the relevant single and double deletion mutants (see Table 1). Transformations were according to Koukaki et al. (2003). Oligonucleotides used are listed in Table S1.

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2.2. Phylogenetic analysis

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Fungal sequences at JGI and NCBI showing significant similarity (>24%) to AcpA and Jen1p transporters were searched and retrieved using standard blastp. The great majority of dikarya possess related sequences, whereas primitive fungi only show a patchy conservation of AcpA- or Jen1-like sequences. Multiple sequence alignment was performed with M-Coffee (http://tcoffee.crg.cat/apps/tcoffee/do: mcoffee; Wallace et al., 2006) and curation of the alignment with trimAI online version (Capella-Gutierrez et al., 2009). Maximum likelihood phylogenetic analysis was performed with Phyml (Guindon et al., 2010) and SH-like approximate likelihood-ratio test (aLTR) was used as statistical test to support branches. Both trimAI and Phyml were used through the Phylemon2 online interface (http://phylemon2.bioinfo.cipf.es/index.html). Trees shown include sequences from all Aspergillus species and representatives from all major dikarya taxa, including all AcpA- or Jen1p-like yeast transporters known to be involved in carboxylic acid transport.

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2.3. Transport assays

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[1-14C]-acetic acid uptake in A. nidulans was assayed in germinating conidiospores, at the polarity maintenance stage, as described in

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Krypotou and Diallinas (2014). Briefly, 107 conidiospores germinating for 4 h at 37 °C, at 130 rpm, in liquid MM supplemented with 1% (w/v) glucose as carbon source and urea as nitrogen source were used. Conidiospores were collected by centrifugation for 5 min at 4000 rpm, resuspended in 4 ml of MM and distributed in 75 ll aliquots that are incubated in a heat-block at 37 °C for 5 min prior to uptake. Transport reaction was started by addition of 25 ll of a radioactive stock prepared at 4.5 nCi nmol 1 (specific activity, s.a.) and stopped by addition of an equal volume of ice-cold 1000-fold concentrated unlabeled substrate. The conidiospores were collected by centrifugation at 13,000 rpm for 5 min, washed once with icecold MM and resuspended in 1 ml of scintillation buffer. For each time point, measurements were performed in triplicate and in three independent experiments. Initial velocities were measured at 1 min of incubation with 30 lM [1-14C]-acetate, pH 6.0, since the initial uptake rate was linear for at least 100 s. The effect of inhibitors was assayed through the addition of non-labelled compounds at the concentrations specified in figure legends to the radiolabelled mixture. Radioactivity was measured in a Packard Tri-Carb 2200CA liquid scintillation counter with disintegrations per minute correction. Background uptake values were corrected by subtracting values obtained in the simultaneous presence of 1000-fold excess of non-radiolabelled substrate. All transport assays were carried out in at least three independent experiments, with three replicates for each concentration or time point. Standard deviation was <20%. Radiolabelled [1-14C] acetate, sodium salt (59 mCi mmol 1) was purchased from Amersham Biosciences, USA. Uptake measurements were also performed using [1,4-14C]-succinic acid (s.a. 1.8 nCi nmol 1), purchased from Moravek Biochemicals, and with D,L[U-14C]-lactic acid (s.a. 1.8 nCi nmol 1), purchased from Amersham Biosciences, at the desired concentration as described for acetic acid. The transport kinetics best fitting the experimental initial uptake rates and the kinetic parameters were determined by a computerassisted non-linear regression analysis (using GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com). In experiments investigating the effect of internally accumulated ammonium on [1-14C]-acetate (30 lM) uptake, cells were preloaded with non-radiolabelled ammonium for 5 min, 37 °C, washed three times in ice-cold MM followed by radiolabelled acetate uptake measurements (Diallinas, 2013). Incubation with non-radiolabelled acetate was used as a control in this experiment. To investigate the efflux of methyl-ammonium, [14C]-methylamine hydrochloride (20 lM), was allowed to accumulate for 5 min at 37 °C, cells were washed three times with ice-cold MM and then loaded with 10 mM of non-labelled acetate and incubated for 5 min at 37 °C, stopped on ice and washed again (Diallinas, 2013). Cells with no loading of non-radiolabelled acetate were used as a control of efflux measurements. Radioactivity accumulated in the cells and in the supernatant was measured as described in Diallinas (2013). Radiolabelled [14C]-Methylamine hydrochloride (57 mCi mmol 1) was obtained from Amersham Pharmacia Biotech.

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3. Results

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3.1. AcpA activity is regulated in response to acetate

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Cells of isogenic acpA+ and acpAD strains were grown in glucose (1.0%, w/v) or fructose (1.0%, w/v) as carbon sources, and in ammonium (10 mM) or urea (5 mM) as nitrogen sources. Acetate uptake measurements were performed in germinating conidiospores, at pH 6.0 (acetic acid predominantly in the deprotonated form as the pKa of acetic acid is 4.75 at 25 °C), as described in Section 2. No significant difference was observed between glucose and fructose or between ammonium and urea (Fig. 1A). The presence of

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sodium acetate at 11 mM as the sole carbon source leads to a decrease in acetate uptake to approximately 40% of that found in the absence of acetate (Fig. 1A). As this reduction was somehow unexpected, given previous reports that acetate induces acpA transcription (Robellet et al., 2008), we wondered whether acetate at the concentration used as a carbon source was toxic to A. nidulans. One possible explanation for this reduced activity would be the accumulation of acetyl-CoA produced by metabolism leading to toxic effects of acetate as is the case for some acetate utilisation mutants (Flipphi et al., 2014). To verify if acetate at the concentration used was toxic, acetate uptake measurements and cell viability were performed at two different germination stages (results not shown). No significant differences were observed in cell viability in the presence or absence of sodium acetate (11 mM). Thus, the apparently reduced AcpA activity observed in media containing solely acetate as a carbon source could not be attributed to reduced viability of cells. We further tested the nature of the acetate effect on AcpA, by adding acetate to glucose-grown cultures. More specifically, cells were grown on glucose for 4 h after which sodium acetate (11 mM) was added to the media, and transport activity was monitored at 0, 1, 2 and 3 h after acetate addition. Clear induction of AcpA transport activity was observed upon addition of acetate, reaching a maximum of 10-fold after 2 h (Fig. 1B). No significant acetate transport was detected in an isogenic acpAD strain, even upon addition of acetate, showing that acetate uptake is mediated by AcpA. This result is in agreement with induction of acpA transcription upon acetate addition in lactose grown cells, shown previously by Robellet et al. (2008). Thus, although acetate reduces the apparent transport activity of AcpA when supplied as the sole carbon source (Fig. 1A), it leads to induction when added for shorter periods in glucose-grown cells (Fig. 1B). The above results suggest that AcpA apparent expression can be up- or down-regulated in response to acetate in the growth medium. This observation might be linked to the fact that acetate is a weak carbon source and, in addition, a potentially toxic metabolite. This means that prolonged growth in acetate might lead to carbon starvation coupled with adverse cytotoxic effects. Both carbon starvation and accumulation of potentially toxic substrates are phenomena that lead to down-regulation of transporters. In particular, carbon starvation leads to transcriptional repression and endocytic turnover of transporters, whereas accumulation of toxic substrates also leads to endocytic turnover (Lauwers et al., 2010). Similar phenomena might control AcpA expression in response to acetate presence in the growth medium. Indeed, Northern analysis shows that the levels of acpA are inversely correlated with acetate concentration and at higher acetate concentrations the reduction is most probably a consequence of acetate toxicity (Robellet et al., 2008). We could not, unfortunately, investigate whether acetate also elicits endocytic turnover of AcpA, as a fluorescent tagged version of this protein did not reach the plasma membrane regardless at which terminus the GFP was fused (results not shown).

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3.2. AcpA is the major acetate transporter during germination

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Previous acetate uptake assays were performed solely at a specific stage during conidiospore germination, coincident with the end of the so-called isotropic growth phase in A. nidulans. Here, we followed the activity of AcpA during the entire period of germination (0–5 h), germling generation (5–7 h) and young mycelia development (8 h) (Fig. 2), in order to identify the stage at which AcpA-mediated transport is maximal in relation to background acetate transport due to other secondary transporters. Germination was carried out in minimal media (MM), supplemented with urea (5 mM) as the sole nitrogen source and glucose (1%, w/v) as the sole carbon source, at 37 °C.

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Fig. 2. AcpA is the major acetate transporter during germination. Uptake of radiolabelled acetate (30 lM) in germinating conidiospores of acpA+ and acpAD strains grown in minimal media supplemented with glucose (1%, w/v) and urea (5 mM) as carbon and nitrogen sources, respectively. 0 h corresponds to resting conidiospores, 1–2 h to the isotropic growth phase, 5 h coincides with germ tube emergence, 6–7 h corresponds to germling and 8 h to young mycelia. At each time point a 10 ml sample of the culture was removed for the uptake assay (see Section 2). Each data point represents the mean ± SD (n = 3). SD was always less than 15%.

Fig. 1. AcpA activity is regulated in response to acetate. (A) Uptake of radiolabelled acetate (30 lM) in A. nidulans germinating conidiospores grown for 4 h in minimal media supplemented with different carbon or nitrogen sources. Each data point represents the mean ± SD (n = 6). Gluc – 1% (w/v) glucose; Fruct – 1% (w/v) fructose; NH4 – 10 mM ammonium tartrate; Urea – 5 mM urea; Acet – sodium acetate 11 mM. (B) Uptake of 30 lM of labelled acetate in A. nidulans acpA+ and acpAD. Cells were grown on glucose for 4 h and collected at 0, 1, 2 and 3 h upon a pulse of 11 mM of sodium acetate. Each data point represents the mean ± SD (n = 6). SD (standard deviation) was always less than 15%.

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Low AcpA-mediated acetate uptake was present in resting conidiospores, in contrast with what has been reported for other A. nidulans transporters specific for amino acids, purines or pyrimidines, which show no uptake in dormant conidia (Tazebay et al., 1997; Amillis et al., 2004). A significant increase in AcpA activity was detected at the end of the isotropic growth phase (2–3 h of germination), and continued to increase until the onset of germ tube appearance (5 h). Immediately after that, AcpA activity diminished rapidly in germlings and young mycelia (Fig. 2). A similar dramatic drop of transporter expression in the transition from conidiospores to germlings has been observed for other transporters (Amillis et al., 2007; Vlanti and Diallinas, 2008). The maximum activity of AcpA detected during the isotropic growth phase is in accordance with what had been observed for other solute transporters in A. nidulans. It has been proposed that developmental activation of transporters during this stage serves as a nutrient sensing and scavenging mechanism, which will eventually adapt uptake and metabolic systems to specific growth media (Amillis et al., 2004). Importantly, a low, AcpA-independent, acetate transport system, just evident during germination of the acpAD strain, increased significantly upon mycelium development after 8 h of

growth (Fig. 2). This observation predicted the existence of at least one other, unknown, acetate transporter, which is activated during vegetative growth.

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3.3. AcpA mediates low-efficiency uptake of other monocarboxylic acids

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Previous studies showed that an acpAD mutant manifests a growth defect at low acetate concentrations, more stringent with increasing pH of the medium, conditions under which the protonated form (acetic acid) is limited (Robellet et al., 2008). We confirmed this phenotype, by further showing that the AcpA permease is essential for the use of acetate as the sole carbon source, mostly visible at 37 °C, pH 6.8–8.0. Additionally, we verified that AcpA contributes to acetate toxicity when spores are germinating in the presence of glucose at 25 °C (Fig. 3A). These growth tests provide physiological evidence that AcpA mediates significant acetate transport in vivo. In addition however, an extended growth test on different carboxylic acids as carbon sources, also suggested that AcpA might contribute to low-capacity uptake of other carboxylic acids, such as pyruvic, propionic, butyric or benzoic acid (see Fig. 3B). To further characterize the specificity of AcpA, mono-, di- and tricarboxylates were tested for their capacity to inhibit AcpA-mediated uptake of radiolabelled acetate. The inhibitors were tested at a concentration 1000-fold over acetate. The monocarboxylates propionic, benzoic, formic and butyric acid inhibited the uptake of acetate by >70% (Fig. 3C). Other monocarboxylic acids, such as salicylic, lactic and pyruvic acid, and some di- or tri-carboxylates had a lower inhibitory effect (10–55%), while maleic, succinic and malic acid (dicarboxylates) had no inhibitory effect on acetate uptake. Overall, these results point to a clear and specific preference of AcpA for several short-chain monocarboxylic acids. We estimated the Ki values for the monocarboxylates that inhibited acetate uptake at a significant level (Table 2). The Km of acetic acid at pH 6.0 (0.16 ± 0.01 mM of acetate) is presented for comparison in Table 2, a value in the same order of magnitude as the one estimated by Robellet et al. (2008) at pH 6.8. Our results show that AcpA binds all tested monocarboxylates with affinity constants, ranging from 1.44 mM to 16.89 mM, with the following affinity order: propionate > butyrate, formate, benzoate. Thus,

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Table 2 Apparent Ki values (mM) of AcpA for acetic, benzoic, butyric, formic and propionic acids.

+

Fig. 3. Substrate specificity profile of AcpA. (A) Growth test of isogenic acpA and acpAD strains on acetate as a carbon source (upper panel) or as a toxic metabolite. (B) Growth test of isogenic acpA+ and acpAD strains on various short-chain monocarboxylic acids at 37 or 25 °C, at pH 5.0, 6.8 and 8.0. Carbon sources used were glucose (1%, w/v); acetic acid (87 mM); butyric, pyruvic, benzoic and lactic acids at 50 mM; malic, succinic and propionic at 60 mM. Images were overcontrasted in order to visualize subtle differences in conidiation. (C) Competition of AcpA-mediated acetate transport by monocarboxylates (see Section 2). Each data point represents the mean ± SD (n = 3). SD was always less than 15%.

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AcpA, is a high-affinity, high-capacity, transporter for acetate with an estimated Km of 0.16 mM acetate at pH 6.0 (compared to 0.23 mM, estimated by Robellet et al. (2008) at pH 6.8). AcpA

Inhibitor

Ki (mM)

Acetic acid Benzoic acid Butyric acid Formic acid Propionic acid

0.16 ± 0.01 16.89 ± 2.12 9.25 ± 1.01 12.06 ± 3.29 1.44 ± 0.13

may also mediate low-affinity uptake of several other monocarboxylate ions.

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3.4. AcpA is not involved in ammonium or methyl-ammonium efflux

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Ady2, the S. cerevisiae orthologue of AcpA, has been reported to transport acetate into the cell (Paiva et al., 2004), but also to be involved in ammonium transport in the reverse direction (Palková et al., 2002). Thus, we also assessed the possible role of AcpA in ammonium transport. The addition of ammonium to the reaction mixture at high concentrations (50 and 100 mM) promoted an increase in acetate uptake of up to 2-fold in acpA+ cells, while no effect was detected in acpAD (Fig. 4A). This effect was independent of whether we used ammonium chloride, sulfate or tartrate, suggesting that it is the addition of ammonium ion that increases the uptake (not shown). We then assessed acetate transport kinetics in the presence of ammonia (50 mM) revealing no significant alterations in the affinity (Km) for acetate, and only a slight increase in the maximal apparent transport rate (Fig. 4B). To further understand the effect of ammonium on AcpA activity, we also investigated whether the intracellular accumulation of ammonium would elicit an increase in AcpA-mediated acetate transport. No difference of AcpA-mediated acetate uptake was recorded upon pre-loading with ammonium for 5 min (Fig. 4C), suggesting that there is no acetate exchange with ammonia. These results suggest that AcpA does not act as NH+4 exporter, at least in the conditions tested. How could the stimulating effect of external ammonium on AcpA be explained? Given that this phenomenon is immediate (1–5 min), we should exclude any effect of ammonium on protein steady state levels or post-translational modification of AcpA. One possible explanation would be that ammonium might chemically affect the protonation state of AcpA. It should be stressed that, even for Ady2, no direct evidence was found for ammonium efflux, which has been speculated on the basis of increased expression of the gene in the presence of ammonium and a correlation between ammonia production and Ady2 activity (Palková et al., 2002). Previous studies have also shown that cells expressing the three ATO genes (including Ady2, also named ATO1) are more resistant to the toxic effect of methyl-ammonium (Palková et al., 2002). This was taken as another piece of indirect evidence for Ady2 involvement in ammonium/ methyl-ammonium efflux. To test whether this is also the case for AcpA, we first examined the effect of methyl-ammonium on growth of acpA+ and acpAD isogenic strains (Fig. 4D). We did not detect any difference related to methyl-ammonium toxicity in the two strains. Subsequently, we employed direct uptake assays with radiolabelled methylamine (methyl-ammonium ion). No difference was observed between the amount of methyl-ammonium in both the supernatant and the pellet (A. nidulans cells) of acpA+ or acpAD cultures (Fig. 4E). This confirms that AcpA is not involved in methyl-ammonium, and very probably ammonium, uptake or efflux.

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We performed a blastp search for secondary acetate transporters, other than AcpA. For that we used as in silico probes the

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Fig. 4. Investigation of the role of AcpA in ammonium or methyl-ammonium transport. (A) Transport rates of radiolabelled acetate (30 lM) uptake in isogenic acpA+ and acpAD strains in the absence or presence of unlabelled ammonium tartrate (NH+4), at the concentrations indicated (5.0–100.0 mM). (B) Transport kinetics of AcpA-mediated radiolabelled acetate uptake in acpA+ cells strains in the absence or presence of unlabelled ammonium tartrate (50 mM NH+4). Each data point represents the mean ± SD (n = 3). Estimated Km and Vm values are shown in the insert. (C) Transport rates of radiolabelled acetate (30 lM) uptake in acpA+ cells pre-incubated with ammonium tartrate or acetate (NH+4), at the concentrations indicated, for 5 min. as described in Section 2. (D) Growth tests of acpA+ and acpAD strains on minimal media supplemented with urea as the nitrogen source and glucose as carbon source (pH 6.8). Methyl-ammonium hydrochloride (MeNH4) was added to this media to a final concentration of 10 mM. Cells were grown for 3 days at 37 °C. (E) Uptake or efflux of radiolabelled methyl-ammonium (CH3NH3Cl) in acpA+ or acpAD cells. In brief, germinated conidiospores, preloaded with [14C]-methyl-ammonium (20 lM), were loaded with 10 mM acetate and subsequently allowed 5 min at 37 °C, before measuring radioactivity in the supernatant and in the cells (for details see Section 2).

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AcpA sequence and the Jen1p transporter of S. cerevisiae. The rationale for this choice is that the S. cerevisiae AcpA homologue, known as Ady2p, and the Jen1p transporter, are the two main transporters responsible of acetate transport. Ady2p is specific for acetate, propionate and formate, whereas Jen1p is specific for lactate, pyruvate, propionate and acetate (Casal et al., 2008). It should be noted that, Ady2p and Jen1p belong to evolutionary and structurally distinct transporter families, the first predicted to be composed of six transmembrane a-helical segments (TMS) with six annotated members encoded in the A. nidulans genome (Flipphi et al., 2006),

while the latTer has the more standard topology of 12 TMS and a common structural fold with members of the well-studied Major Facilitator Superfamily (Yan, 2013). We identified genes encoding two Ady2p/AcpA homologues (AN1839 and AN7317) and two Jen1p homologues (AN6703 and AN6095) in the genome of A. nidulans (Flipphi et al., 2006; Robellet et al., 2008), which have not been characterized previously. We performed a standard phylogenetic analysis in order to identify their origin and divergence in fungi (see Section 2), which provided us with some clues regarding their function and specificity. Fig. 5A

Please cite this article in press as: Sá-Pessoa, J., et al. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.02.010

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and B shows collapsed phylogenetic trees of AcpA-like and Jen1plike proteins, respectively, in dikarya fungi. AcpA-like proteins are well conserved in the dikarya analyzed with a few exceptions. Proteins very similar to AcpA (>74% identity in Eurotiomycetes) form a distinct clade, which includes members from all 21 species of Aspergillus with known genomes and most ascomycetes. A second clade, which includes AN1839, is on average 56% similar to the AcpA proteins and is also well, but not absolutely, conserved in Aspergillus sp. or other dikarya (e.g. not present in A. fumigatus, Aspergillus oryzae or Aspergillus flavus). The functionally characterized yeast homologues (S. cerevisiae, and Yarrowia lipolytica) form a distinct clade with more or less equal evolutionary distance from the AcpA and AN1839 clades (50% identity). The third A. nidulans AcpA-like protein, AN7317, belongs to a distinct clade that includes few sequences (only 7 Aspergillus sp. out of the 21 with known genomes have a very similar sequence) and is significantly more distant to the AcpA, AN1839 or the yeast clades (28–33% identity). The basidiomycetes analyzed also possess AcpA-or AN1839-like sequences, whereas similar sequences show a patchy distribution in several primitive fungi (non dikarya). Overall, AcpA and AN1839 belong to two groups of proteins that are well conserved in the species included in this study, suggesting an essential role in fungi. In contrast, the very patchy distribution of AN7317-like sequences or other clades points to a non-essential function. In agreement with the absolute conservation of members of the AcpA group, transcriptomics in A. nidulans (Flipphi et al., 2009; Sibthorp et al., 2013) have shown that AcpA is highly expressed under most conditions tested (nitrate or ammonium as N source, complete media, 4 h N starvation), with a significant drop only under C starvation or prolonged N starvation (72 h). The drop of AcpA expression under C starvation explains the drop in acetate uptake, which we detected after prolonged growth on acetate as a sole carbon source (see Fig. 1A). Low transcription of AN1839 was also detected in transcriptomic analysis under most condition tested, with a significant increase under C starvation or prolonged N starvation. Finally, no transcript was detected, under all conditions tested, for AN7317 (Sibthorp et al., 2013). Jen1-like proteins are present in most dikarya analyzed (Fig. 5). The two A. nidulans paralogues, AN6703 and AN6095, are 39.7% and 30.4% identical with Jen1p of S. cerevisiae. In addition, they conserve the sequence NXX[S/T]HX[S/T]QDXXXT (Soares-Silva et al., 2007) and other specific residues necessary for short-chain carboxylic acid binding and transport (Soares-Silva et al., 2011). The great majority of Aspergillus species have two Jen1-like paralogues, one similar to AN6095 and the other to AN6703. The absolute (AN6703) or nearly absolute (AN6905) conservation of two Jenlike proteins in Aspergilli (Fig. 5) suggests that both have an essential role. Our analysis shows that the group of AN6095-like proteins (subsequently called JenA-like proteins, see later) diverged first in the evolution of fungal Jen proteins, whereas the AN6703 (subsequently called JenB) group is closer to that made up by the yeast Jen1 and Jen2 transporters. Given that yeast Jen1p and Jen2p are specific for either mono- or di-carboxylates, our analysis suggests this specificity distinction occurred in yeast and that AN6095/JenA might be a more promiscuous transporter, being able to recognize all types of short-chain carboxylates, including mono-, di-carboxylates, or even tri- or longer carboxylates. Similarly, as the AN6703/ JenB group is also an out-group of the Jen1/Jen2 clade, it might also be a promiscuous transporter. As will be shown below, this is indeed the case. 3.6. AN1839 contributes to the uptake of acetate while AN6703 and AN6095 contribute to the uptake of other monocarboxylates We genetically constructed, using a standard reverse genetic methodology, null mutants of all novel genes encoding putative

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acetate or other short-chain carboxylic acid transporters, as well as combinations with each other and with acpA (see Section 2). Fig. 6 shows an extensive growth profile of all constructed strains related to short-chain carboxylic acids. Despite the marginal differences of growth of the different mutant stains, a careful observation was suggestive for the function and specificity of the relevant transporters analyzed. First, AN1839 is very likely a secondary acetate transporter since the double acpAD AN1839D mutant shows marginally reduced growth compared to the single acpAD mutant on acetate (mostly visible at pH 6.8–8.0, Fig. 6). In addition, AN1839, together with AcpA, appears to be involved in the uptake of butyric acid (mostly visible at pH 5.0). Second, AN6095 contributes to lactic, pyruvic, malic and succinic acid uptake. Third, AN6703 contributes to a malic and succinic acid uptake. Fourth, there is a pH-dependent shift in the apparent capacity for transport of dicarboxylic acids (malic and succinic) of AN6095 and AN6703; AN6095 contributes to their uptake mostly at pH 8.0, whereas AN6703 does so at lower pH values. In other words, AN6095 seems to be specific for both mono- and dicarboxylic acids, especially at basic pH, whereas AN6703 seems specific for dicarboxylic acids. However, as will be shown later, both AN6095 and AN6703, can contribute to the uptake of mono- and dicarboxylic acids, but with different efficiencies. This partially contrasts the case in yeasts, where Jen1-like and Jen2-like transporters are strictly specific for either monocarboxylic acids or dicarboxylic acids, respectively (Casal et al., 2008). Based on the phylogenetics and the analysis of null mutants we propose to rename these genes/proteins as follows: AN1839 as AcpB, AN6095 as JenA, and AN6703 as JenB. The remaining gene/ protein (AN7317) was tentatively named AcpC, based solely on its sequence similarity with AcpA and AcpB. The lack of any apparent function of AcpC related to carboxylic acid transport is also in line with its lack of evidence for transcription in transcriptomics analysis (Sibthorp et al., 2013) and its patchy conservation in fungi, suggestive of gene loss. Recently, we provided evidence for the existence of putative proteins resembling genuine purine transporters, that seem to correspond to non-functional unstable polypeptides, probably on their way to extinction (Krypotou et al., in press).

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3.7. AcpB contributes to acetate transport mostly in mycelia

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Given that growth tests suggested that AcpB might be a secondary active transporter and that low AcpA-independent acetate uptake was detected in young mycelia, we proceeded to measuring directly the possible contribution of AcpB (AN183), but also of AcpC (AN7317), in acetate transport, by uptake assays in mycelia of acpAD, acpAD acpBD, acpAD acpCD and acpAD acpBD acpCD strains (Fig. 7). Significant AcpB-mediated acetate transport was measured in the acpAD and acpAD acpCD strains. Very low acetate transport was measured in acpAD acpBD and acpAD acpBD acpCD strains. This probably reflects passive diffusion or a very low activity of an unknown transporter. Thus, AcpB is confirmed to be the main secondary acetate transporter expressed in mycelia, whereas AcpC has no apparent function related to acetate transport.

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3.8. JenA and JenB are transporters specific for short-chain carboxylic acids, but not for acetate

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To access the function and specificity in respect to mono- versus dicarboxylic acid transport of JenA and JenB, we performed uptake assays of radiolabeled acetic, lactic and succinic acids in germinating conidiospores of appropriate relevant null mutants (jenAD acpAD, jenBD acpAD, jenAD jenBD acpAD). No difference in acetic acid uptake was obtained among the three strains, confirming that JenA and JenB do not catalyse acetate transport (not shown).

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Fig. 5. Phylogenetic analysis of AcpA-like and Jen1p-like putative carboxylic acid transporters. (A) Collapsed phylogenetic tree of AcpA-like fungal proteins. (B) Collapsed phylogenetic tree of Jen1p-like fungal proteins. AcpA and Jen1p homologues from Coemansia reversa were used as an outgroup. Protein IDs are shown next to species name and are from JGI or AspGD. Species shown in the trees are as follows. Eurotiomycetes: Aspergillus sp, Neosartorya fischeri, Talaromyces stipitatus; Pucciniomycotina: Puccinia graminis; Ustilaginomycotina: Ustilago maydis; Agaricomycotina: Cryptococcus neoformans; Pezizomycetes: Ascobolus immerses; Orbiliomycetes: Monacrosporium haptotylum; Dothideomycetes: Cochliobolus heterostrophus; Lecanoromycetes: Xanthoria parietina; Leotiomycetes: Sclerotinia sclerotiorum; Sordariomycetes: Podospora anserina, Neurospora crassa; Xylonomycetes: Xylona heveae; Saccharomycotina: Saccharomyces cerevisiae, Kluyveromyces lactis, Candida albicans, Yarrowia lipolytica; Taphrinomycotina: Schizosaccharomyces pombe, Taphrina deformans.

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Transporter-mediated lactic acid and succinic acid uptake was detected in both jenAD and jenBD single mutants, whereas the double jenAD jenBD mutant showed non-significant uptake of these acids, other than passive diffusion (Fig. 8). Thus JenA and JenB are the major transporters mediating uptake of lactic acid and succinic acid, JenA being a little more efficient for lactic acid (higher Vm), whereas JenB showing higher affinity for succinic acid (lower Km). Table 3 summarizes the kinetic characteristics of JenA and JenB. In conclusion, both transporters recognize mono- and di-carboxylates, but with different transport kinetics.

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AcpA has been described previously as a permease essential for the uptake and use of acetate as sole carbon source (Robellet et al., 2008). In this work, we show that AcpA is a high-affinity, high-capacity, acetate transporter, which can also recognize monocarboxylates with medium or low affinity and transport them with low capacity. We further show that AcpA acts as the major acetate transporter during conidiospore germination and also operates at lower levels in mycelia. The expression profile of AcpA differs from other transporters studied in A. nidulans so far with respect to two aspects. First, low AcpA activity is detected in resting conidiospores, suggesting a possible role in spore maintenance or homoeostasis. In

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A. nidulans and A. fumigatus, enzyme activities of the glyoxylate pathway (for instance AcuD, the isocitrate lyase) have been detected in conidiospores, suggesting a role related to the oxidation of fatty acids to carbohydrates in supplying nutrients to germlings (Ebel et al., 2006; Osherov et al., 2002). Both the isocitrate lyase and AcpA are activated by the same transcriptional regulator FacB (Ebel et al., 2006; Robellet et al., 2008). Furthermore it should be noted that the enzyme FacA (acetyl-CoA synthase) can produce acetate from the acetyl-CoA intracellular pool (end-product of the oxidation of fatty acids) (Takasaki et al., 2004). Therefore, early activity of AcpA in resting conidiospores might be justified since transporters are efficient scavengers that can supply metabolites avoiding the cost of biosynthesis. In addition, the non-repressibility of AcpA by a primary carbon source such as glucose, shows that this transporter serves cellular needs for acetate accumulation other than a supplier of a carbon source. It should also be noted that AcpA activity drops at the transition from isotropic to polarized growth, in agreement with transcriptome analysis showing that germination leads to a shift from fermentative metabolism to respiration in A. fumigatus (Lamarre et al., 2008). AcpA recognizes, and possibly transports, propionic, formic, benzoic or butyric acid. These however, are recognized with approximately 7- (propionate) to 70- (formate, benzoate, butyrate) fold lower affinities. At least in the case of propionate, we obtained evidence from growth tests that propionate is transported into the

Please cite this article in press as: Sá-Pessoa, J., et al. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.02.010

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Fig. 6. Involvement of putative AcpA-like and Jen1p-like proteins in transport. (A) Growth tests of single, double and triple null mutants of AcpA- and Jen1p-like proteins on various carboxylic acids as sole carbon sources. Cultures were incubated for 3 days at 37 °C or for 6 days at 25 °C. Carbon sources used were glucose (1%, w/v), acetic acid (87 mM); butyric, pyruvic, benzoic and lactic acids at 50 mM; malic, succinic and propionic at 60 mM. Images were over-contrasted in order to visualize subtle differences in conidiation. Wt and acpAD growth test image rows are repeated from Fig. 3B for direct comparison with the double and triple mutants.

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cells and that it probably binds to a single substrate-binding site, as does acetate. The observation that monocarboxylic acids, but not dicarboxylic acids, gain access to the binding site of AcpA suggests that the substrate binding site of this transporter is narrow and binds only small molecules with one carboxylate group. Ady2p, the homologue of AcpA in S. cerevisiae, shares a similar specificity profile, suggesting an architecturally similar binding site. On the other hand, the homologous bacterial transporter SatP from Escherichia coli (34% identity with AcpA) is an acetate-succinate transporter (Sá-Pessoa et al., 2013), accepting both mono- and dicarboxylic acids, and thus might have a slightly different substrate-binding site in relation to its fungal homologues. Given the proposed role of Ady2p in ammonium efflux (Palková et al., 2002), we also investigated whether AcpA can act similarly.

Although we detected a stimulation of AcpA-mediated acetate uptake in the presence of ammonium at high concentrations (50 and 100 mM), growth tests and efflux assays showed that AcpA is not directly related to ammonium or methyl-ammonium efflux. As ammonium at high concentrations reduces the pH of the medium (Freitas et al., 2007), one can hypothesize that this might have a direct effect on the function of AcpA by altering its protonation state (Schönichen et al., 2013) or an indirect effect on the plasma membrane allowing increased acetate uptake by simple diffusion. In the second part of this work we looked for secondary acetate transporters and in particular we wanted to identify the gene(s) encoding the residual acetate transport activity in the acpFD mutant, particularly in mycelia. The obvious candidates tested were the two paralogues of AcpA, called AcpB and AcpC, and the

Please cite this article in press as: Sá-Pessoa, J., et al. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.02.010

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two homologues of S. cerevisiae Jen1p, called JenA and JenB. Indeed, one of them, AcpB, was found to be responsible for the residual acetate transport in mycelia of acpFD or acpA acpCD mutants. No function was found for AcpC, in agreement with lack of detectable transcription and minimal evolutionary conservation in fungi. Finally, JenA and JenB were shown to act as monocarboxylic and dicarboxylic acids transporters, but were not involved in acetate transport. Interestingly, JenA, which seems to diverge first in the evolutionary tree, before the evolution of transporters specific for either mono- or di-carboxylic acids, recognizes equally well both monocarboxylic and dicarboxylic acids, supporting that specificity Table 3 Kinetic parameters of JenA and JenB for lactic and succinic acids. The kinetic parameters were estimated by subtracting the values obtained for the triple mutant acpAD jenAD jenBD.

Fig. 7. AcpB contributes to acetate transport mostly in mycelia. Uptake of radiolabelled acetate (30 lM) in acpAD, acpAD acpBD, acpAD acpCD and acpAD acpBD acpCD strains in young mycelia.

Km (mM)

Vm (nmol min

JenA

Lactic acid Succinic acid

0.57 ± 0.21 0.21 ± 0.12

2.59 ± 0.38 0.98 ± 0.11

JenB

Lactic acid Succinic acid

0.97 ± 0.08 0.33 ± 0.07

0.97 ± 0.06 0.43 ± 0.03

1

107 conidiospores)

Fig. 8. JenA and JenB are transporters specific for short-chain carboxylic acids. Initial uptake rates of radiolabelled lactic acid (A) and succinic acid (B) as a function of substrate concentration in acpAD jenBD (d), acpAD jenAD (j), acpAD jenAD jenBD (N). Each data point represents the mean ± SD of 3 independent experiments (n = 9).

Please cite this article in press as: Sá-Pessoa, J., et al. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.02.010

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arises from promiscuity (Harms and Thornton, 2013; Atkins, 2014). On this line, JenB, which is closer to the yeast Jen1/2 specific transporters, seems to have reduced promiscuity compared to JenA, being more efficient for di-carboxylic rather than monocarboxylic binding, as judged by growth tests (Fig. 6), but also in relation to binding affinities (Fig. 8). Finally, a novel, biotechnologically exploitable, aspect concerning JenA and JenB function is the observation that pH changes can shift specificity from monocarboxylic to dicarboxylic acids and vice versa. JenA is more specific for dicarboxylates at pH 8.0, whereas JenB does so at lower pH values (see Fig. 6).

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Brock and Buckel (2004).

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Acknowledgments

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We are very grateful to Emila Krypotou for her help in the phylogenetic analyses. We are grateful to Dr. Velot for his kind gift of the two strains acpA+ and acpAD indispensable for this work. This work was supported by FEDER, through POFC – COMPETE and by Portuguese National Funds from ‘‘FCT – Fundação para a Ciência e a Tecnologia’’, in the scope of the projects PEst-C/BIA/UI4050/ 2011 and PEst-OE/BIA/UI4050/2014. JSP [SFRH/BD/61530/2009] received a fellowship from the Portuguese government from FCT through POPH and FSE.

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Please cite this article in press as: Sá-Pessoa, J., et al. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.02.010