Phenotypic characterization of the Golgi-localized AkrA

Based on a NCBI BLASTp search (http://www.ncbi.nlm.nih.gov/BLAST/), we identified a putative ortholog of NFAT in A. nidulans, AkrA (AN5824.4, Accession: XP_663428.1), which encodes a putative palmitoyltransferase. However, it showed low identity (less than 20%) or similarity (less than 30%) to mammalian NFAT based on full-length sequences. Interestingly, a bioinformatic analysis revealed that the promoter region contains a putative calcineurin-dependent-response-element (CDRE-like) motif. As shown in Fig 1A, we identified a CDRE-like sequence at 398 bp (akrA, AN5824.4), upstream of this gene’s start codon [26,27]. These data suggest that AkrA may be a component of the calcium signaling machinery. To further explore the function of the akrA gene and its relationship to calcineurin, the full-length deletion strain was constructed by homologous gene replacement employing a self-excising recyclable cassette that contains an AfpyrG gene as a selectable marker. Diagnostic PCR analysis of the resulting strain ΔakrA confirmed the homologous replacement (S1A Fig). We also generated ΔakrAΔcnaA double mutants through genetic crosses (the cnaA gene encodes the catalytic subunit of calcineurin). The ΔakrA mutant produced smaller colonies compared to that of the parental wild-type strain, when grown on minimal medium. In comparison, the ΔcnaA mutant exhibited severe growth defects on minimal medium. Moreover, the double mutant had a smaller colony size and underwent less conidiation than the single mutants (Fig 1B). These results suggest that akrA and cnaA may have different functions in A. nidulans. Therefore, the double deletion mutant exacerbates the growth defects on minimal medium.

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Fig 1. Identification of AkrA in A. nidulans.

A. Alignment of Crz1/CrzA DNA-binding sites. CDRE consensus sequences 1 and 2 correspond to those described in previous studies. A CDRE-like sequence was identified at 398 bp (akrA, AN5824.4) upstream of its respective start codon. B. The colony morphologies of TN02A7 (WT), ΔakrA, ΔcnaA and ΔakrAΔcnaA strains grown on minimal media at 37°C for 2.5 days. C. The TN02A7 (WT) and ΔakrA strains were incubated at 37°C for 2.5 days on minimal medium in the presence or absence of 1 mM EGTA or 20 mM CaCl₂. D. The pattern of conidiation and hyphal branching in TN02A7 (WT), ΔakrA and revertant strains. Images were taken with a stereo microscope after culturing colonies for 2.5 days on solid non-inducing medium and culturing mycelial balls for 24 h in liquid non-inducing medium, respectively.

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We next tested whether low external calcium conditions could affect the colony phenotype in the akrA deletion mutant. When conidia were spot inoculated onto the solid minimal medium containing the calcium chelator EGTA and were allowed to grow at 37°C for 2.5 days, the ΔakrA mutant exhibited increased EGTA sensitivity compared to the parental wild-type strain. As shown in Fig 1C, the akrA deletion exhibited markedly reduced conidial formation and colony growth under low-calcium conditions. Since, mutants of the HACS components have been previously shown to exhibit similar defects under low calcium conditions [28–30], we next examined whether AkrA was a potential novel HACS component. To determine whether the defects in the ΔakrA mutant could be rescued by high extracellular calcium, we inoculated ΔakrA mutant conidia on minimal medium supplemented with 20 mM Ca²⁺. We found that the colony diameter of the ΔakrA mutant was restored almost to the same diameter of the parental wild-type strain by the addition of extracellular calcium (Fig 1C), indicating that exogenous calcium could completely rescue the colony growth defect caused by AkrA loss. We further examined conidiation in the ΔakrA mutant in a calcium-limited environment (i.e. in the presence of EGTA) with a stereomicroscope (Fig 1D left panels). The results showed that the vegetative mycelia from the parental wild-type strain were capable of producing numerous conidia under low-calcium conditions. In contrast, conidiation was almost completely abolished in the ΔakrA mutant on minimal media supplemented with EGTA (1 mM) (Fig 1D left panels). In submerged liquid culture, the wild-type strain displayed robust polarized hyphal growth around the margins of mycelial balls, whereas the ΔakrA mutant showed smooth margins around small mycelial balls (Fig 1D right panels). Consistently, the ΔakrA mutant had a significantly reduced biomass, germination rate, and colony size compared to the parental strain on minimal media (S3 Fig). Moreover, ectopically expressed akrA was able to completely rescue these defects in the akrA deletion strain (Fig 1D), establishing that these phenotypes were specific to the loss of akrA. In addition, we deleted the akrA homolog gene in A. fumigatus. Similar to the ΔakrA phenotypes in A. nidulans, the ΔAfakrA mutant displayed hypersensitivity to the low calcium conditions, and its phenotypic defects could be rescued by high extracellular calcium (S2 Fig). Thus, these data are consistent with AkrA being involved in calcium uptake especially in a calcium-limited environment.

To further confirm and assess the localization and the molecular mass of AkrA, we generated a conditional expression allele, alcA(p)::GFP-akrA, referred to here as ZYA09 (S1B Fig). In this conditional allele, akrA expression was assumed to be regulated by the carbon source, as it was not induced by glucose, induced by glycerol, and overexpressed to high levels by L-threonine [31]. To determine whether this conditional allele behaved as predicted, we inoculated the ZYA09 strain in liquid media for 18 h, which promoted induction, non-induction or overexpression. As expected, the akrA mRNA level was approximately 20-fold higher when grown in overexpressing medium compared to that grown in non-inducing medium, which was 12-fold higher than that in inducing medium (S4B Fig). Moreover, the conditional strain ZYA09 displayed an identical phenotype to the parental wild-type strain when grown on the inducing or the overexpressing media, indicating that the fusion GFP-AkrA protein was functional and that the assumed akrA over-expression had no detectable effects in A. nidulans. In comparison, when grown on the non-inducing medium, the conditional allele alcA(p)::GFP-akrA exhibited an identical phenotype to the ΔakrA mutant, confirming a consistent phenotype for the loss of AkrA and for the knock-down of AkrA (Figs 2A and 1C). Western blotting showed a band at approximately 110 kDa in the GFP-AkrA strain grown under inducing or overexpressing conditions using an anti-GFP antibody but no such a band appeared in the parental wild-type strain or the conditional allele (ZYA09) under the non-inducing condition (Fig 2B). These results indicate that the molecular mass of AkrA is approximately 80 kDa because GFP is a 27 kDa protein.

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Fig 2. Phenotypic characterization of Golgi-localized AkrA.

A. The phenotypic characterization of akrA under control of the alcA(p) conditional promoter. The colony images show corresponding strains grown on the non-inducing medium (RE::akrA), inducing medium (EX::akrA) and overexpressing medium (OE::akrA) at 37°C for 2.5 days. B. Western blot analysis indicated a fusion protein of GFP-AkrA was detected with a predicted size of approximately 100 kDa by using an anti-GFP antibody. GFP-AkrA non-inducing and GFP-AkrA inducing represent alcA(p)::GFP-akrA grown in liquid non-inducing medium and inducing medium, respectively. Anti-actin antibody against actin was used as an internal control of loading. C. Colocalization of GFP-AkrA and the GEs marker mRFP-PH^OSBP. A strain carrying transgenes expressing the two fluorescent reporters was imaged using GFP and mRFP specific filter sets. The yellow color in the merged image shows the co-localization. Bar, 5 μm.

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Microscopic examination showed that the AkrA-GFP localization pattern resembled that of the Golgi previously reported in A. nidulans [32]. To confirm this we generated the strain ZYA13 by genetically crossing the alcA(p)::GFP-akrA strain ZYA09 with the MAD2013 strain in which the late Golgi marker (gpdA^mini::mRFP-PH^OSBP), consisting of the pleckstrin homology domain of the human oxysterol binding protein (PH^OSBP) fused to mRFP was included [33,34]. Spores of the ZYA13 strain were incubated in non-inducing medium at 37°C for 10 h and were then shifted to the overexpression medium for 6 h. Microscopic examination of the young germlings produced under these conditions showed the majority of GFP-AkrA proteins colocalized with mRFP-PH^OSBP late Golgi marker (Fig 2C).

The DHHC motif is required for AkrA function

Because the bioinformatic analysis showed that AkrA contains a conserved DHHC motif required for its palmitoylation activity [19–21], we next investigated whether the DHHC motif was required for the normal function of AkrA under low calcium conditions. We first constructed a C-terminal AkrA truncation lacking the region from the DHHC motif through to the stop codon by homologous gene replacement (Fig 3A). The colony phenotype of the truncation mutant was similar to that resulting from the complete deletion of the akrA gene when grown in minimal medium plus EGTA, indicating that the DHHC motif is required for AkrA function (Fig 3B). To rule out the possibility that a loss of function in the truncated mutant might result from a conformational change that prevented a true reflection of the function of the DHHC motif, we performed site-directed mutagenesis. Since Cys⁴⁸⁷ in the DHHC motif has previously been shown to be crucial for palmitoyl transferase activity, we therefore mutated Cys⁴⁸⁷ to Ser⁴⁸⁷ in the DHHC motif (Fig 3A) [35,36]. Consequently, we found that the C487S site-mutated DHHS fragment could not rescue the defect of the akrA deletion mutant under either the control of a native promoter (native(p)::akrA^C487S) or a GPD promoter (GPD(p)::akrA^C487S) (Fig 3B). In comparison, the wild-type akrA gene completely rescued the growth defects in the akrA deletion recipient strain. To confirm that these fusion cassettes were transcribed in the transformant, we performed quantitative real-time PCR to verify the akrA mRNA levels. The results showed that both the GPD and native promoters induced normal akrA mRNA expression, even though the mRNA expression level under the control of the GPD promoter was higher than that with the native promoter (S4D and S4E Fig), indicating that the AkrA-DHHS cassettes were fully transcribed. Next, we generated Flag-tagged AkrA and the site mutated AkrA^C487S strains to further confirm the expression of the AkrA protein. As shown in Fig 3C, the predicted bands on a Western blot were observed clearly, suggesting that both Flag-AkrA and Flag-AkrA^C487S proteins were fully expressed in vivo. In addition, the Flag-tagged AkrA^C487S strain displayed an identical phenotype to that of the Flag-untagged (native(p)::akrA^C487S) mutant, suggesting that the Flag tag could not phenotypically change the function of the targeted protein AkrA (Fig 3B and 3D). These data suggest that the growth defect caused by akrA deletion was closely associated with the Cys⁴⁸⁷ site in the DHHC motif.

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Fig 3. The DHHC motif is required for the function of AkrA.

A. The predicted secondary structure of AkrA. It contains five predicted transmembrane domains, six ankyrin repeat sequences mapping to the NH₂-terminal hydrophilic domain, and a DHHC-CRD sequence located between transmembrane domains 3 and 4. A hydrophobicity plot using the SOSUI program (http://harrier.nagahama-i-bio.ac.jp/sosui/) predicted a secondary amino acid structure for AkrA. The C-terminal truncated mutant and mutation site of the AkrA-C487S were labeled as indicated by the arrow. B. The colony morphology and conidiation pattern of TN02A7 (WT), ΔakrA, akrAΔC, native(p)::akrA^C487S and GPD(p)::akrA^C487S grown on solid minimal media in the presence or absence of 1mM EGTA or 20 mM CaCl₂, respectively, at 37°C for 2.5 days. C. Western blot analysis of total protein extracts of TN02A7 (WT), Flag-AkrA and Flag-AkrA^C487S strains probed with anti-Flag antibody. Anti-actin antibody against actin was used as an internal control of loading. D. Growth phenotype of indicated strains grown on solid minimal media in the presence or absence of 1mM EGTA or 20 mM CaCl₂, respectively, at 37°C for 2.5 days.

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AkrA functions independently of previously identified HACS components

Because the loss of akrA caused a similar defect phenotype to that of deletion mutants of the HACS components cchA and midA under the low calcium conditions, we hypothesized that AkrA forms a complex with CchA or MidA to perform its function. To assess whether AkrA physically interacts with CchA or MidA, we performed yeast two-hybrid assays. We cloned the cDNA fragments corresponding to the cytosolic C-terminus of cchA and the full-length cDNA of midA, respectively. They were then amplified and cloned into the pGADT7 vector, which contains the GAL4 DNA-AD and the LEU2 marker. In addition, a full-length cDNA of akrA was cloned into the pGBKT7 vector, which contains the GAL4 DNA-BD and TRP1 marker. As a result, some small colonies of pGBKT7-akrA with pGADT7-cchA were obtained, and there was no detectable growth of colonies of pGBKT7-akrA with pGADT7-midA under the high stringency screening conditions compared to the positive colonies of pGADT7-T and pGBKT7-53, which showed robust growth (S4A Fig). These data suggest that AkrA and MidA do not directly interact, and that AkrA and CchA might weakly and transiently interacted.

We next investigated the functional interaction(s) between AkrA and CchA and between AkrA and MidA by a genetic phenotypic analysis. The ΔakrAΔmidA, ΔakrAΔcchA double mutants were generated by genetic crossing. As shown in Figs 4A and S6, phenotypic defects in colony size and conidiation were exacerbated in the double mutants compared to the parental single mutants, especially in the presence of EGTA. Notably, the growth retardation of the ΔakrAΔmidA and ΔakrAΔcchA double mutants under low calcium conditions was reversed by the addition of 20 mM calcium to the minimal medium. These results suggest that AkrA, CchA, and MidA are all required under the calcium-limited condition, but may have some non-overlapping roles in growth. To determine whether overexpression of cchA could rescue the ΔakrA defects under the low calcium condition, we crossed ΔakrA (ZYA02) and alcA(p)::GFP-cchA (ZYA11) to generate the ZYA12 strain. Real-time PCR verified that the mRNA level of cchA in ZYA12 was approximately 15-fold higher in the overexpressing medium than in the inducing medium when cultured for 18 h (S4C Fig). However, overexpression of cchA did not rescue the ΔakrA defects under low calcium conditions (Fig 4B).

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Fig 4. Relationship between AkrA and the CchA, MidA and PmrA.

A. Colony morphology comparison for the indicated strains grown on solid minimal media in the presence or absence of 1 mM EGTA or 20 mM CaCl₂ at 37°C for 2.5 days. B. Colony phenotypes of the indicated strains at a series of 2 μL 10-fold dilutions derived from a starting suspension of 10⁶ conidia/mL grown on solid inducing medium (upper panels) or solid overexpressing medium (lower panels) in the presence or absence of 1mM EGTA at 37°C for 2.5 days.

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Previous studies have demonstrated that pmr1, which encodes a Ca²⁺/Mn²⁺ P-type ATPase and is involved in Ca²⁺ homeostasis, localizes to the Golgi in yeast [37]. In A. nidulans, ΔpmrA had no discernible effect on fungal physiology, but the cells were hypersensitive to low extracellular calcium [38]. To investigate the link between AkrA and PmrA, we crossed the ΔakrA and ΔpmrA mutants. Surprisingly, the double mutant had no detectable defect when grown in minimal medium compared to the ΔakrA strain, which had a reduced-colony size (Fig 4A). These data suggest that the pmrA deletion suppressed the ΔakrA growth defect. However, when cultured on minimal medium with 1 mM EGTA, the double mutant showed an exacerbated growth retardation phenotype compared to the parental single mutants. In addition, the phenotypic defects of ΔakrAΔpmrA were completely suppressed by the addition of 20 mM calcium. These results suggest that AkrA and PmrA may operate together in regulating cellular calcium homeostasis in a reverse way.

AkrA mediates the extracellular calcium-induced [Ca²⁺]_c transient

Previous studies with yeast reported that Cch1 and Mid1 mutations reduced calcium uptake and affected [Ca²⁺]_c accumulation under both stimulating and non-stimulating conditions [5,39–41]. We monitored the extracellular calcium-induced [Ca²⁺]_c changes in living cells of A. nidulans wild type and mutant strains in which we expressed codon-optimized aequorin [42–44]. When treated with 0.1 M CaCl₂, the [Ca²⁺]_c concentration in wild type cells transiently increased from a resting level of approximately 0.1 μM to a peak concentration of 1.2 μM (Fig 5). In comparison, cchA or midA mutants showed a reduction of 17 ± 11% or 25 ± 12% in the [Ca²⁺]_c amplitudes, respectively, under the same stimulating conditions. Surprisingly, the decrease in the [Ca²⁺]_c amplitude in akrA mutants was much larger than that observed in the HACS mutants. The [Ca²⁺]_c amplitudes were decreased as follows: 53 ± 13% in the akrA deletion strain ZYA02, 54 ± 9% in the DHHC truncated mutant ZYA15, and 55 ± 8% in the site-mutated native(p)::akrA^C487S mutant ZYA16. These data suggest the significant reduction in calcium influx due to the loss of AkrA is mediated by the DHHC motif and, in particular, the cysteine residue within the DHHC motif.

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Fig 5. Extracellular Ca²⁺-induced [Ca²⁺]_c transients in akrA mutants.

[Ca²⁺]_c responses in the wild type and indicated mutant strains following a stimulus of high external calcium (0.1 M CaCl₂) with the peak [Ca²⁺]_c amplitudes expressed as a percentage of that of the wild-type. The bar graph shows the peak [Ca²⁺]_c concentrations of the indicated strains after treatment with CaCl₂. The basal [Ca²⁺]_c resting level is indicated by the line (approximately 0.1 μM in these experiments), **p<0.01. Values represent averages of six wells and error bars represent SD (n = 6).

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The [Ca²⁺]_c amplitude in the ΔpmrA mutant exposed to the 0.1 M CaCl₂ stimulus was similar to that of the parental wild-type strain, which is different from that previously reported for yeast [45–47], suggesting that other Ca²⁺-ATPases may compensate for the loss of PmrA function in response to the extracellular calcium stimulus. However, loss of pmrA in the akrA deletion background was able to recover the decreased [Ca²⁺]_c amplitude in the akrA mutant to a similar level as that in the parental wild-type strain in response this extracellular calcium stimulus, indicating that the perturbation of calcium homeostasis induced by AkrA could be rescued by loss of pmrA.

The protein palmitoylation inhibitor 2-bromopalmitate (2-BP) is a palmitate analog that blocks palmitate incorporation into proteins [48,49]. To determine whether inhibition of palmitoyl transferase activity influences calcium influx into the cytoplasm, we measured the [Ca²⁺]_c amplitude of the wild type pre-incubated in 2-BP (20 μM) for 2 h. Following this drug treatment, the amplitude of the [Ca²⁺]_c increase following stimulation with 0.1 M CaCl₂ was significantly reduced by approximately 40% of the untreated cells in response to stimulation with 0.1 M CaCl₂ (Fig 5). These data suggest that the inhibition of palmitoyl transferase activity can significantly block calcium influx.

Loss of AkrA abolishes [Ca²⁺]_c responses to ER or plasma membrane stress

Activation of Ca²⁺ channels, calmodulin, calcineurin and other factors is necessary for the long-term survival of cells undergoing ER stress, and during this process the HACS components, CchA and MidA, are required for Ca²⁺ influx from the extracellular environment [41,50,51]. To verify whether AkrA is involved in the calcium influx response during ER stress, we measured the influence of the ER-stress agents, tunicamycin (TM) and dithiothreitol (DTT) on [Ca²⁺]_c. When the parental wild-type strain was treated with 5 μg/mL tunicamycin, we observed an immediate transient increase in [Ca²⁺]_c with an amplitude of 0.60 ± 0.03 μM (Fig 6B). In comparison, the [Ca²⁺]_c amplitude in the ΔcchA mutant (but not the ΔmidA mutant) in response to tunicamycin was decreased by approximately 32 ± 6%, suggesting that the loss of CchA but not MidA mediates the ER stress-induced calcium influx in A. nidulans. Furthermore, in response to tunicamycin treatment the [Ca²⁺]_c amplitude decreased by 40 ± 5%, 34 ± 8% and 34 ± 6% in the ΔakrA, akrAΔC, native(p)::akrA^C487S mutants, respectively. We next examined the [Ca²⁺]_c response after addition of DTT, another agent causing ER-stress. 10 mM DTT induced a rapid increase in [Ca²⁺]_c which peaked at approximately 0.40 μM in the wild-type and ΔmidA strains, but the [Ca²⁺]_c amplitudes decreased by approximately 40% in the ΔakrA (36 ± 10%), akrAΔC (37 ± 7%), and native(p)::akrA^C487S (36 ± 8%) mutants, and by 15 ± 9% in the ΔcchA mutant (S7 Fig). These data suggest that CchA, but not MidA, influences the ER stress-induced calcium influx in A. nidulans, which is different from that previously reported in yeast [41,51]. Furthermore, loss of AkrA, or mutations in its DHHC significantly decreased the ER stress-induced calcium influx. We further tested whether the amplitude of the [Ca²⁺]_c increase in response to tunicamycin was dependent on the extracellular calcium concentration. We found that there was no significant change when mycelia were cultured in media with or without 5 mM calcium (S8A Fig). In contrast, exposure of cells to 1 mM EGTA prior to tunicamycin treatment completely abolished the increase in [Ca²⁺]_c in the ΔakrA, akrAΔC and native(p)::akrA^C487S mutants, but not in the parental wild-type, ΔcchA or ΔmidA strains (Fig 6A). Similar data was obtained when we used the more selective, calcium chelator BAPTA (S9 Fig). These data suggest that intracellular calcium stores contribute to the transient increase in [Ca²⁺]_c induced by agents causing ER stress.

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Fig 6. AkrA regulates the [Ca²⁺]_c transient induced by ER stress following tunicamycin treatment.

A. [Ca²⁺]_c responses in the wild type and indicated mutant strains to 5 μg/mL tunicamycin pretreated for 10 min with the calcium chelator EGTA (1 mM). The peak [Ca²⁺]_c amplitudes are expressed as a percentage of that of the wild-type. The bar graph shows the peak [Ca²⁺]_c concentrations of the indicated strains after treatment with EGTA and Tunicamycin (TM) (right panel). The basal [Ca²⁺]_c resting level is indicated by the line (approximately 0.08 μM in these experiments), **p<0.01. In each experiment, values represent averages of six wells and error bars represent SD (n = 6). B. [Ca²⁺]_c responses in the wild type and indicated mutant strains to 5 μg/mL TM. The bar graph shows the peak [Ca²⁺]_c concentrations of the indicated strains after treatment with TM (right panel). The basal [Ca²⁺]_c resting level is indicated by the line (approximately 0.1 μM in these experiments), **p<0.01.

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Because azole antifungal drugs induce plasma membrane stress [13,14,52], we next compared the differences in the [Ca²⁺]_c transient between wild-type and relevant mutant strains after treatment with the azole antifungal agent itraconazole (ITZ), which is currently used as a primary antifungal drug in the clinic. In all the tested mutants and the wild-type strain, the [Ca²⁺]_c resting levels were similar at approximately 0.05 μM. After addition of 1 μg/mL ITZ to the medium, all strains responded with a transient increase in [Ca²⁺]_c (Fig 7B). However, all the akrA defective mutants exhibited significantly lower increases in [Ca²⁺]_c compared to their parental wild-type strain: the amplitudes of the [Ca²⁺]_c transients were reduced by 36 ± 11% in the ΔakrA, 29 ± 10% in the akrAΔC, 24 ± 8% in the native(p)::akrA^C487S and 27 ± 8% in the ΔcchA mutants, respectively, compared to that of the parental wild-type strain. In marked contrast to these mutants, the ΔmidA mutant exhibited a similar [Ca²⁺]_c amplitude in response to ITZ as observed in the wild-type strain. In addition, the amplitude of the ITZ-induced [Ca²⁺]_c elevation increased when mycelia were cultured in media containing 5 mM CaCl₂ (S8B Fig). We next examined whether the [Ca²⁺]_c transient induced in response to ITZ was dependent on external calcium or internal calcium stores. We exposed hyphal cells to media supplemented with EGTA (1 mM) prior to ITZ treatment, and found that [Ca²⁺]_c transients were dramatically abolished in all the ΔakrA mutants, whereas the [Ca²⁺]_c transients in the wild type, and the ΔcchA and ΔmidA mutants, were still observed (Fig 7A). Similar data were obtained when we used the calcium chelator BAPTA (S9 Fig). These data indicate that the loss of AkrA or disruption of its DHHC motif in the absence of extracellular calcium completely block calcium influx after treatment with chemicals that induce ER or plasma membrane stress from both extracellular and intracellular sources. Furthermore, both extracellular calcium and intracellular calcium stores play roles in generating these [Ca²⁺]_c transients induced by these stress treatments.

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Fig 7. akrA regulates the [Ca²⁺]_c transient induced by plasma membrane stress following antifungal azole treatment.

A. [Ca²⁺]_c responses in the indicated strains to ITZ (1 μg/mL) pretreated for 10 min with the calcium chelator EGTA (1 mM). The peak [Ca²⁺]_c amplitudes are expressed as a percentage of that of the wild-type. The bar graph shows the peak [Ca²⁺]_c concentrations of the indicated strains after treatment with EGTA and ITZ (right panel), **p<0.01. The basal [Ca²⁺]_c resting level is indicated by the line (approximately 0.06 μM in these experiments). In each experiment, values represent averages of six wells and error bars represent SD (n = 6). B. [Ca²⁺]_c responses to ITZ (1 μg/mL) in the indicated strains. The bar graph shows the peak [Ca²⁺]_c concentrations of the indicated strains after treatment with ITZ (right panel), **p<0.01. The basal [Ca²⁺]_c resting level is indicated by the line (approximately 0.09 μM in these experiments).

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The cysteine residue of the DHHC motif is required for AkrA palmitoylation

Our evidence above indicates that the cysteine residue in the DHHC motif of AkrA is involved in regulating the calcium response to high extracellular calcium-, ER- and plasma membrane-stress. To test whether the cysteine residue of DHHC is required for AkrA palmitoylation, we set up an acyl-biotin exchange (ABE) chemistry assay to detect palmitoylation in potential target proteins based on selective thioester hydrolysis by hydroxylamine (HA) (Fig 8A). Compared to the control, the treatment of hydroxylamine combined with N-ethylmaleimide (NEM) (which blocks free sulhydryls), efficiently enriches palmitoylated proteins. Subsequent treatment with HA cleaves the thioester bond between palmitate and cysteine residues, exposing bound thiols, which are then covalently linked to HPDP-biotin. The controls were protein samples not treated with HA. Lastly, the biotinylated proteins were bound to streptavidin agarose, washed with buffer, and eluted by cleavage of the cysteine-biotin disulfide linkage following by SDS-PAGE. Several previous reports have suggested that the process of palmitoylation involves in a two-step mechanism in which palmitoyl transferase is auto-acylated by itself to create an intermediate followed by the transfer of the palmitoyl moiety to its substrate [53,54]. Therefore, to investigate whether the cysteine residue in the DHHC motif is responsible for AkrA auto-acylation, we used the ABE assay to detect whether AkrA palmitoylates itself [20]. As shown in Fig 8B, when HA was present, Flag-AkrA can be clearly detected with an anti-Flag antibody. However, a site-directed mutation of the cysteine residue in the DHHC motif and the parental wild-type strain pre-cultured with 2-bromopalmitate (2-BP) completely abolished palmitoylation of AkrA, which resulted in no signal being detected in the enriched pamitoylated proteins. These results indicate that AkrA is able to be auto-acylated and the cysteine residue in the DHHC motif is required for this process. In addition, we found that treatment with 2-BP (24 h, 50 and 100 μM) virtually abolished the Golgi localization of GFP-labelled AkrA (Fig 8D) and resulted in a similar defective growth defect phenotype to the ΔakrA mutant on minimal medium (S10 Fig).

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Fig 8. The cysteine residue in the DHHC motif, is correspondingly required for AkrA palmitoylation.

A. A schematic diagram of the acyl-biotin exchange (ABE) assay. ① Blocking the free sulfhydryls with N-ethylmaleimide (NEM); ② Cleavaging the thioester bonds with or without hydroxylamine (HA); ③ Biotinylating the palmitoylated proteins with HPDP-biotin; Lastly, ④ Enriching the biotinylated proteins bound to streptavidin agarose (SA). B. Flag-AkrA and Flag-AkrA^C487S were detected by Western blotting with anti-Flag antibodies using the ABE assay, treated or not with 100 μM 2-bromopalmitate (2-BP). Hydroxylamine (HA) was used to specifically cleave S-acyl groups revealing sulfhydryl groups, which were subsequently labeled with biotin. Samples were then bound to streptavidin beads. For the negative control HA was substituted by Tris. Anti-actin antibody was used as an internal control of loading. A band was detected in the +HA treated sample, indicating that it was bound to an acyl group via a thioester linkage confirming that it is auto-acylated. However, no signal was detected for Flag-AkrA^C487S and 2-BP treatment samples and therefore they are not auto-acylated. C. Western blot analysis indicated a fusion protein of GFP-AkrA^C487S was detected with a predicted size of approximately 100 kDa by using an anti-GFP antibody. D. GFP-AkrA and GFP-AkrA^C487S localization was assessed after culturing for 18 h in liquid induced medium supplemented with or without the indicated concentration of 2-BP. Localization within the Golgi was less distinct as punctate structures in the GFP-AkrA^C487S strain compared with that in the wild-type and its localization within the Golgi was completely abolished after 2-BP treatment. Bar, 2 μm. E. Total proteins from wild type and ΔakrA strains subjected to the ABE assay with (HA+) or without (HA-) hydroxylamine treatment. The samples were then electrophoresed by SDS-PAGE and detected by silver nitrate staining.

https://doi.org/10.1371/journal.pgen.1005977.g008

We constructed another alcA(p)::GFP-akrA^C487S mutant and confirmed by Western blotting (Fig 8C) to further check whether site directed mutagenesis of the Cys⁴⁸⁷ in the DHHC motif disrupted the normal localization of AkrA in the Golgi. The GFP-AkrA^C487S was less distinctly localized in the punctate Golgi structures characteristic of wild-type GFP-AkrA and some appeared to be localized in the cytoplasm (Fig 8D). These data collectively suggest that the cysteine residue in the DHHC motif of AkrA and the palmitoylation activity are closely associated with AkrA auto-acylation, which is required for normal AkrA localization and palmitoylation.

To further explore palmitoylated protein substrates specifically mediated by AkrA, total proteins of the wild-type and ΔakrA strains were treated and analyzed using the ABE chemistry assay combined with liquid chromatograpy-mass spectrometry (LC-MS) for comparative proteomics (Fig 8E). Using this approach, 334 proteins were identified as potential AkrA substrates in the parental wild-type strain because they were completely absent in the ΔakrA strain. As shown in Table 1, AkrA belonged to one of the AkrA-mediated pamitoylated substrates suggesting it is able to auto-acylate itself. Among the palmitoylated protein candidates identified, Yck2, Lcb1, Ras2, Cdc48 and Pab1 have been previously identified as palmitoylated proteins in S. cerevisiae but only Yck2 has been characterized as an Akr1 substrate [20,55–57]. These data indicated that the ABE chemistry assay combined with LC-MS was a valid approach to identify proteins palmitoylated by AkrA and it also indicated that A. nidulans may palmitoylate some of the substrates previously reported in S. cerevisiae. In our study we notably identified the following protein substrates palmitoylated by AkrA: a vacuolar Ca²⁺-ATPase Pmc1 homolog (AN5088.4); a P-type ATPase Spf1 homolog (AN3146.4) involved in calcium homeostasis [58]; a putative V-type H⁺-ATPase Vma5 homolog (AN1195.4) that has been linked to Ca²⁺-ATPase function [59], and three uncharacterized proteins (AN8774.4, AN3420.4 and AN2427.4), the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner [53]. These results provide strong evidence that the AkrA protein regulates [Ca²⁺]_c homeostasis in A. nidulans by palmitoylating these protein candidates. Other candidate substrates of AkrA that we identified included the P450 enzymes, Cyp51A (Erg11A), Cyp51B (Erg11B) and Erg5 homologs, which are all involved in ergosterol biosynthesis and azole resistance. Thus AkrA may influence the azole resistance by these biosynthetic enzymes.

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Table 1. Selected A. nidulans palmitoylated proteins.

https://doi.org/10.1371/journal.pgen.1005977.t001

AkrA mediates the [Ca²⁺]_c transient in response to high extracellular calcium stress independently of the CchA/MidA complex

Deletion of the akrA gene exhibited marked growth and conidiation defects under low calcium conditions, which is similar to the defects caused by mutations in the CchA/MidA HACS [28–30]. In addition, the akrA deletion conferred increased sensitivity to Li⁺, Na⁺, K⁺, Mg²⁺, but slightly increased resistance to the cell wall disrupting agents compared to the parental wild-type strain (S5 Fig). Moreover, the ΔakrAΔcchA and ΔakrAΔmidA double mutants exacerbated the ΔakrA defects under calcium-limited conditions, suggesting that AkrA may have independent functions to those of the CchA-MidA complex. AkrA localized to trans Golgi structures (Fig 2C), while the CchA-MidA complex probably localizes to the plasma membrane as reported for yeast [40,66,67]. In addition, results from the Y2H assays (S4A Fig) suggested that there were no direct, or only very weak, interactions between AkrA and CchA and between AkrA and MidA. Nevertheless, the [Ca²⁺]_c transient in the ΔakrA mutant had a much lower amplitude (approximately 53 ± 13% lower) than the wild-type control following treatment with a high extracellular calcium stress stimulus, suggesting that the loss of AkrA reduced calcium influx into the cytoplasm. In contrast, loss of CchA and MidA caused a 25% decrease in the [Ca²⁺]_c amplitude in response to this treatment with high external calcium, consistent with the results from previous studies on yeast cells lacking either Cch1 or Mid1, which exhibited a low calcium uptake [5,39–41]. The akrA deletion also had a bigger impact on inhibiting calcium influx in response to ER stress than observed in the ΔcchA and ΔmidA mutants. Overall our data suggests that AkrA regulates calcium uptake from the external medium as well and its release from intracellular Ca²⁺ stores through a pathway that is independent of the previously identified CchA/MidA HACS as shown in Fig 9. PmrA is an A. nidulans homolog of yeast Pmr1, which is a P-type Golgi Ca²⁺/Mn²⁺ ATPase responsible for Ca²⁺ transport into the Golgi and widely accepted as responsible for Ca²⁺ efflux from the cytoplasm into the Golgi to regulate calcium signaling and homeostasis and prevent calcium toxicity. Loss of Pmr1 function in budding yeast is believed to inhibit the return of [Ca²⁺]_c to its resting level following stimulus-induced [Ca²⁺]_c increases [37,45–47]. In contrast, our data showed that the pmrA deletion in A. nidulans exhibited no significant change in the calcium signature following a high extracellular calcium stress stimulus compared with the wild-type strain, suggesting that other paralogs of pmrA (e.g. other Ca²⁺-ATPases) may compensate or play more important roles in returning the elevated [Ca²⁺]_c back to its resting level. Surprisingly, loss of pmrA alleviated the decreased response of the ΔakrA mutants to the external calcium stimulus, resulting in the amplitude of the [Ca²⁺]_c increase of the double mutant ΔpmrAΔakrA being almost back to the normal level of the wild type. Thus deletion of PmrA reverses the effects of the AkrA deletion in regulating calcium influx following extracellular calcium stress. The lower amplitude of the [Ca²⁺]_c increase of the ΔakrA mutant in response to the high extracellular calcium stimulus indicate that AkrA and its pamitoylated targets play a role in mediating the calcium influx into the cytoplasm and then PmrA may store cytoplasmic calcium into Golgi. When both PmrA and AkrA were absent, the increase in [Ca²⁺]_c following extracellular calcium stimulation was back to almost the normal level in the wild-type (Fig 5). This suggests that the [Ca²⁺]_c increase in the ΔpmrAΔakrA double mutant following treatment with high extracellular calcium is compensated by some other unknown component(s) of the calcium signaling/homeostatic machinery. Furthermore, our data (Fig 4A) showed that loss of pmrA suppressed the colony growth defect of ΔakrA mutants, providing further evidence to support interactive regulatory roles of PmrA and AkrA in A.nidulans.

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Fig 9. A working model of how AkrA function regulates [Ca²⁺]_c homeostasis in A. nidulans.

AkrA protein mediates [Ca²⁺]_c homeostasis by palmitoylating protein candidates labeled by Palm: a putative P-type ATPase Spf1 homolog, a calcium ion transport Vma5 homolog and three uncharacterized proteins, the transcripts of which are induced in response to extracellular calcium stress in a CrzA-dependent manner in A. nidulans.

https://doi.org/10.1371/journal.pgen.1005977.g009

Previous studies have verified that exposure of fungi to ER or plasma membrane stress stimulates store-operated calcium influx through the HACS to promote fungal cell survival [13,14,41,50–52]. Consistent with previous studies, in A. nidulans we observed a transient increase in [Ca²⁺]_c after treatment with the ER-stress agents tunicamycin (TM) or dithiothreitol (DTT). The ΔcchA mutant exhibited reduced [Ca²⁺]_c amplitudes by 32 ± 6% and 15 ± 9% upon treatment with TM or DTT, respectively (Figs 6 and S7). In contrast, we did not detect a change in the [Ca²⁺]_c response to the ER stress agents in the ΔmidA mutant compared to its parental wild-type strain. This suggests that as a complex of CchA and MidA, CchA may have a more predominant role than MidA during the ER stress response. Moreover, the ΔakrA mutant displayed a decreased response to ER and plasma membrane stress inducing drugs, as the [Ca²⁺]_c amplitude of ΔakrA mutants decreased by approximately 36–40% of the wild-type strain following treatment with these drugs (Figs 6 and S7). These data suggest that, in addition to HACS components, AkrA is also involved in ER and plasma membrane stress-induced calcium influx. Moreover, these responses were completely abolished in the ΔakrA mutant but not in the wild-type strain in the presence of EGTA or BAPTA that chelate external calcium. These results indicate that both extracellular calcium and calcium stores contribute to the transient [Ca²⁺]_c changes following ER or plasma membrane stress. Because calcium release from intracellular stores in response to these types of stress was abolished in the akrA mutants (Figs 6, 7 and S9), our results are consistent with AkrA regulating calcium influx across the plasma membrane, which in turn activates the release of calcium from intracellular pools.

Altogether, our results provide the first report that AkrA is a putative palmitoyl transferase in A. nidulans, and it mediates calcium influx in a DHHC-dependent mechanism to perform an essential function in calcium homeostasis/signaling for survival under high extracellular calcium-, ER- or azole antifungal-stress conditions.

Calcium signaling regulators have been previously identified as antifungal target candidates, including FK506, which targets calcineurin [8]. However, most of the fungal homologs of known calcium signaling components in mammalian cells are of proteins also required for mammalian cell growth and metabolism [68]. Thus, potential antifungals against these components may cause side effects in mammalian hosts. The use of drugs that target regulators of posttranslational modification of calcium signaling that show significant differences to their mammalian homologs (e.g. AkrA only exhibits 24.8% identity to the human AkrA homolog HIP14), may circumvent this problem. The potential for developing novel antifungal drugs of this type has been greatly facilitated by our study that has shown a critical link between palmitoylation and calcium signaling.

The [Ca²⁺]_c response is closely associated with the cysteine residue in the DHHC motif which is correspondingly required for AkrA palmitoylation

Previous studies have shown that all AkrA homologs across different species require the DHHC motif to be active and function normally as palmitoyl transferases [69–71]. Three approaches were initially employed to determine AkrA function: deletion of the DHHC motif; site-directed mutagenesis of the cysteine residue in the DHHC motif; and use of a specific palmitoyl transferase analogue inhibitor (2-bromopalmitate), to determine AkrA function [48,49]. Our data from these experiments suggested that the DHHC motif and its cysteine residue are required for the function of AkrA, especially when extracellular calcium is limited. To further test whether the cysteine residue in the DHHC motif, is correspondingly required for AkrA palmitoylation, we used the acyl-biotin exchange (ABE) chemistry assay to detect palmitoylation based on selective thioester hydrolysis by hydroxylamine. Compared to the treatment without hydroxylamine, the newly exposed cysteine residues are disulfide-bonded to a biotin analogue, affinity purified and digested into peptides, leaving the labeled peptides on the affinity beads so that palmitoylated proteins have been enriched. As the ABE chemistry detects palmitoylation through identification of all the thioester linkages. A subsequent Western experiment was used to further confirm palmitoylated proteins by specific antibodies. Consequently, among these enriched palmitoylated proteins, Flag-AkrA was clearly detected with an anti-Flag antibody. Site-directed mutation of the cysteine residue in the DHHC or treatment of the parental wild-type strain with the palmitoyl transferase analogue inhibitor 2-BP completely abolished palmitoylation of AkrA (Fig 8B). Previous studies have demonstrated that although the exact mechanism of S-acylation is not known, palmitoylation of the purified DHHC-CRD palmitoylated proteins zDHHC2, zDHHC3 and yeast Erf2, involves a two-step mechanism, in which the zDHHCs form an acyl-enzyme intermediate (auto-acylation), with the acyl group later transferred to the target protein [53,54]. Our results indicated that AkrA auto-acylated itself before palmitoylating its target proteins. In mammalian cells, any protein that contains a surface-exposed and freely accessible cysteine that has transient access to Golgi membranes is susceptible to palmitoylation. Our data suggests AkrA both auto-acylated itself and palmitoylates target proteins in association with Golgi membranes. Moreover, we found that site directed mutagenesis of the Cys⁴⁸⁷ in the DHHC motif significantly affect normal localization of AkrA in the Golgi. When we treated cells with a specific palmitoyl transferase analogue inhibitor 2-BP, AkrA localization within the Golgi localization was completely lost (Fig 8D), suggesting that the 2-BP treatment not only prevented AkrA auto-acyltation but also prevented the normal subcellular localization of AkrA. The reason for the different localization pattern, if any, caused by the site directed mutagenesis and the treatment of 2-BP as shown in Fig 8D is likely to be due to a side effect of the 2-BP reagent.

In conclusion, our results provide the first report that AkrA is a palmitoyl transferase in A. nidulans, and that it mediates calcium influx in a DHHC-dependent mechanism to perform an essential role in calcium homeostasis to survive high extracellular calcium-, ER- and plasma membrane-stress conditions. A working model of AkrA function in regulating [Ca²⁺]_c homeostasis in A. nidulans is presented in Fig 9. Our findings provide new insights into the link between palmitoylation and calcium signaling that may be of relevance for understanding the mechanistic basis of human PAT-related diseases. Regulators of posttranslational modification in fungi may provide promising targets for new therapies against life threatening fungal diseases.

Strains, media, and cultural conditions

All fungal strains used in this study are listed in S1 Table. Minimal media (MM), and MMPDR (minimal media + glucose + pyrodoxine + riboflavin), MMPDR+UU (minimal media + glucose + pyrodoxine + riboflavin+ uridine + uracil), MMPGR (minimal media + glycerol + pyrodoxine + riboflavin) have been described previously [29,72]. MMPGRT was MMPGR with 100 mM threonine. Fungal strains were grown on minimal media at 37°C, harvested using sterile H₂O and stored for the long-term in 50% glycerol at −80°C. Expression of tagged genes under the control of the alcA promoter was regulated by different carbon sources: non-induced by glucose, induced by glycerol and overexpressed by glycerol with threonine. Growth conditions, crosses and induction conditions for alcA(p)-driven expression were as previously described [73].

Construct design and tagging of AkrA with GFP

In order to generate constructs for akrA null mutant (ΔakrA), the fusion PCR method was used as previously described [74]. Primers used to design constructs are listed in S2 Table. The A. fumigatus pyrG gene in plasmid pXDRFP4 was used as a selectable nutritional marker for fungal transformation. The transformation was performed as previously described [75].

For creating an ΔakrA construct, a 5′ flank and a 3′ flank DNA fragments were amplified using the primers akrA-P1 and akrA-P3, akrA-P4 and akrA-P6, respectively, using genomic DNA (gDNA) of the A. nidulans wild-type strain TN02A7 as the template for PCR. As a selectable marker, a 2.8 kb DNA fragment of A. fumigatus pyrG was amplified from the plasmid pXDRFP4 using the primers pyrG-5’ and pyrG-3’. The three PCR products were combined and used as a template to generate a 4.8 kb DNA fragment using the primers akrA-P2 and akrA-P5. The final PCR product was transformed into a wild-type strain. A similar strategy was used to construct akrA-truncated mutants.

To design the revertant strain construct, a 3.7 kb DNA fragment, which included a 1.2 kb promoter region, a 2.4 kb coding sequence, and a 3′ flank was amplified using the primers primer A and primer D from A. nidulans gDNA. As a selectable marker, a 1.7 kb pyroA fragment was amplified from the plasmid pQa-pyroA using the primers pyro-5’ and pyro-3’. The two PCR products were co-transformed into the ΔakrA strain to produce the revertant strain.

To generate the alcA(p)::GFP-akrA vector, a 1 kb akrA fragment was amplified from the gDNA in the wild-type strain TN02A7 with primers akrA-5’ and akrA-3’ (S2 Table) and then ligated into the plasmid vector pLB01 yielding plasmid pLB-alcA(p)::GFP-akrA which contains GFP-N under the control of the alcA promoter with the N. crassa pyr4 as a marker.

For site-directed mutation, a 3.7 kb akrA DNA fragment with a site directed mutation in which cysteine⁴⁸⁷ was replaced by serine and a selective marker pyroA were co-transformed into the ΔakrA strain to obtain native(p)::akrA^C487S strain. The fragment containing the site mutation was amplified with two steps. First, fragment AB and fragment CD were amplified from A. nidulans gDNA with primers A and B, primers C and D, respectively, and complementary regions contained the desired mutation (cysteine⁴⁸⁷ to serine⁴⁸⁷). Second, using fragment AB and fragment CD as a template, the final 3.7 kb fragment was generated through fusion PCR using primer A and primer D.

The GPD(p)::akrA^C487S and alcA(p)::GFP-akrA^C487S strains were constructed using a similar strategy. In brief, the GPD promoter was amplified with the GPD-5’ and GPD-3’, and 2.4 kb akrA DNA fragment including a 2.4 kb coding sequence, and a 0.5 kb 3’ flanking was amplified with akrA-GPD-5’ and primer D. These two fragments were combined using GPD-5’ and primer D, Lastly, the aboved fusion PCR products and the selective marker pyroA were co-transformed into the ΔakrA strain to obtain the GPD(p)::akrA^C487S strian. For the alcA(p)::GFP-akrA^C487S construction, a 5′ flank and a 3′ flank DNA fragments were amplified from genomic DNA of alc-akrA mutant using the primers alc-up and primer B, primer C and new primer D, respectively. Then the two PCR products were combined and used as a template to generate a 3.9 kb DNA fragment using the primers alc-up and new primer D, and then this fragment was ligated into a plasmid vector yielding the pEA-C487S. The pyroA fragment was amplified from the pQa-pyroA using the primers pyro-cre-5’ and pyro-cre-3’, then recombined into the plasmid pEA-C487S. Finally the plasmid was transformed into the ΔakrA strain to obtain the alcA(p)::akrA^C487S strian.

All N-terminal Flag constructs were designed and fabricated using restriction-free cloning protocols outlined at http://www.rf-cloning.com using PrimerSTAR MAX DNA polymerase (TAKARA, R045A) [76]. Then, N-Flag tagged cassettes and selective marker pyroA were co-transformed into the ΔakrA strain.

For the mutants expressing the codon-optimized aequorin, the plasmid pAEQS1-15 containing codon-optimized aequorin and selective markers pyroA or riboB genes were co-transformed into the indicated mutants. Transformants were screened for aequorin expression using methods described previously [77] and high aequorin expressing strains were selected after homokaryon purification involving repeated plating of single conidia.

Plate assays

For each experiment, at least three replicate plates were used to test phenotypes for each strain. To assess the influence by the extracellular calcium to the colony phenotype, minimal medium was supplemented with 20 mM CaCl₂ or 1 mM EGTA, respectively. The influence of osmotic stress or ionic stress was tested by adding 600 mM NaCl, 600 mM KCl, 10 mM MnCl₂, 400 mM MgCl₂, 400 mM CaCl₂ or 300 mM LiCl into minimal medium, respectively. For the cell wall integrity test, the reagent of 60 μg/mL Calcofluor White or 100 μg/mL Congo Red was added to the minimal medium, respectively. 2 μL of conidia from the stock (1×10⁶ conidia/mL) for indicated strains were spotted onto relevant media and cultured for 2.5 days, at 37°C, and then the colonies were observed and imaged.

Fluorescence microscopy observations

For microscopic observations, conidia were inoculated onto pre-cleaned glass coverslips overlaid with liquid media. To observe co-localization of GFP-AkrA and mRFP-PH^OSBP, strain ZYA13 (S1 Table) was cultured at 37°C for 10 h in non-inducing medium (non-inducing conditions for the alcA(p) driving expression of AkrA) and shifted for 6 h to the inducing medium (in which the alcA promoter was induced) before microscopic observation [34]. Differential interference contrast (DIC) and fluorescence images of the cells were captured with a Zeiss Axio imager A1 microscope (Zeiss, Jena, Germany) equipped with a Sensicam QE cooled digital camera system (Cooke Corporation, Germany). The images were processed with MetaMorph/MetaFluor software (Universal Imaging, West Chester, PA) and assembled in Adobe Photoshop (Adobe, San Jose, CA).

Germination assay

Germination was assessed in liquid non-inducing medium at 37°C with a total number of 10⁶ conidia/mL for each strain in their stationary phase [78]. The percentage rate of germination was measured at 4, 5, 6, 7 and 8 h by microscopic examination. Spores were considered as germinated ones when length of the germ tube was almost equal to the conidium in diameter. For each strain, three replicates of 100 spores were quantified at each time point to determine the germination rate.

Yeast two-hybrid assay

Saccharomyces cerevisiae strain AH109 (Clontech, Palo Alto, CA) was used as the host for the two-hybrid interaction experiments. The analysis was performed using the Matchmaker Library Construction & Screening system (BD Clontech). For strain generation, a cDNA fragment corresponding to the cytosol C-terminus of cchA and the full-length cDNA of midA were amplified and cloned into the pGADT7 vector, which contains the GAL4 DNA-AD and the LEU2 marker (BD Clontech). Full-length cDNA of akrA were used for the pGBKT7 vector (Clotech, Palo Alto, CA).

[Ca²⁺]_c measurement

The strains expressing the codon-optimized aequorin gene were grown on minimal media for 2.5 days to achieve maximal conidiation. 10⁶ spores with liquid media were distributed to each well of a 96-well microtiter plate (Thermo Fischer, United Kingdom). Six wells were used in parallel for each treatment. The plates were incubated at 37°C for 18 h. The medium was then removed and the cells in each well were washed twice with PGM (20 mM PIPES pH 6.7, 50 mM glucose, 1 mM MgCl₂). Aequorin was reconstituted by incubating mycelia in 100 μL PGM containing 2.5 μM coelenterazine f (Sigma-Aldrich) for 4 h, at 4°C in the dark. After aequorin consititution, mycelia were washed twice with 1 mL PGM and allowed to recover to room temperature for 1 h [79,80]. To chelate extracellular Ca²⁺, 1 mM EGTA or 8 mM BAPTA was added to each well 10 min prior to stimulus injection.

At the end of each experiment, the active aequorin was completely discharged by permeabilizing the cells with 20% (vol/vol) ethanol in the presence of an excess of calcium (3 M CaCl₂) to determine the total aequorin luminescence of each culture. Luminescence was measured with an LB 96P Microlumat Luminometer (Berthold Technologies, Germany), which was controlled by a dedicated computer running the Microsoft Windows-based Berthold WinGlow software. Conversion of luminescence (relative light units [RLU]) into [Ca²⁺]_c was done using Excel 2007 software (Microsoft). The relative light units (RLU) values were converted into [Ca²⁺]_c concentrations by using the following empirically derived calibration formula: pCa = 0.332588 (-log k) + 5.5593, where k is luminescence (in RLU) s^-1/total luminescence (in RLU) [77]. Error bars represent the standard error of the mean of six independent experiments, and percentages in the figures represent peak of [Ca²⁺]_c compared to that of the wild-type (100%).

Acyl-biotin exchange (ABE) assay and mass spectrometry

ABE was performed as described previously with some modifications [81]. Briefly, the strain mycelium was ground to a fine powder in liquid nitrogen and resuspended in 5 mL lysis buffer. Samples were incubated for 1 h at 4°C followed by centrifugation at 4°C, 13,000 g to remove insoluble material. 5 mg of protein was incubated overnight with 50 mM N-ethylmaleimide (NEM) at 4°C to reduce proteolysis while allowing free sulhydryls to be blocked. Proteins were precipitated at room temperature using methanol/chloroform. The pellet was resuspended in 200 μL resuspension buffer and the solution divided into two equal aliquots. One aliquot was combined with 800 μL of 1 M fresh hydroxylamine (HA), 1 mM EDTA, protease inhibitors and 100 μL 4 mM biotin-HPDP (Thermo Scientific). As a control the remaining aliquot was treated identically but hydroxylamine (HA) was replaced with 50 mM Tris pH 7.4. Proteins were precipitated and resuspended in 100 μL of resuspension buffer. 900 μL PBS containing 0.2% Triton X-100 was added to each sample, aliquots were removed as a loading control, and the remaining reactions were incubated with 30 μL of streptavidin-agarose beads (Thermo scientific). The streptavidin beads were washed four times with 1 mL PBS containing 0.5 M NaCl and 0.1% SDS. Proteins were eluted by heating at 95°C in 40 μL 2× SDS sample buffer containing 1% 2-mercaptoethanol v/v. Samples were analyzed by silver staining or Western blotting as described below. In some cases, cells were treated with 50 or 100 μM of the palmitoylation inhibitor 2-bromopalmitate (2-BP) before the ABE assay.

For mass spectrometry (MS), total protein (100 μg) extracted from each sample was chemically reduced for 1 h at 60°C by adding DTT to 10 mM and carboxyamidomethylated in 55 mM iodoacetamide for 45 min at room temperature in the dark. Then trypsin gold (Promega, Madison, WI, USA) was added to give a final substrate/enzyme ratio of 30:1 (w/w). The trypsin digest was incubated at 37°C for 16 h. After digestion, the peptide mixture was acidified by 10 μL of formic acid for further MS analysis. After protein digestion, each peptide sample was desalted using a Strata X column (Phenomenex), vacuum-dried and then resuspended in a 200 μL volume of buffer A (2% ACN, 0.1% FA). After centrifugation at 20000 g for 10 min, the supernatant was recovered to obtain a peptide solution with a final concentration of approximately 0.5 μg/μL. 10 μL supernatant was loaded on a LC-20AD nano-HPLC (Shimadzu, Kyoto, Japan) by the autosampler onto a 2 cm C18 trap column. The peptides were then eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μL/min for 4 min, then the 35 min gradient was run at 300 nL/min starting from 2 to 35% buffer B (95% ACN, 0.1% FA), followed by a 5 min linear gradient to 60%, then followed by a 2 min linear gradient to 80%, and maintenance at 80% buffer B for 4 min, and finally returned to 5% in 1 min.

Data acquisition was performed with a TripleTOF 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA). Data was acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 psi, nebulizer gas of 15 psi, and an interface heater temperature of 150. The MS was operated with a RP of greater than or equal to 30,000 FWHM for TOF MS scans. Raw data files acquired from the Orbitrap were converted into MGF files using Proteome Discoverer 1.2 (PD 1.2, Thermo), [5,600 msconverter] and the MGF file were searched. Protein identification was performed by using Mascot search engine (Matrix Science, London, UK; version 2.3.02) against a database containing 13,597 sequences.

Western blotting analysis

To extract proteins from A. nidulans mycelia, conidia from alcA(p)::GFP-akrA and the wild-type strains were inoculated in the liquid inducing medium, then shaken at 220 rpm on a rotary shaker at 37°C for 24 h. The mycelium was ground in liquid nitrogen with a mortar and pestle and suspended in ice-cold extraction buffer (50 mM HEPES pH 7.4, 137 mM KCl, 10% glycerol containing, 1 mM EDTA, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, 1 mM PMSF). Equal amounts of protein (40 μg) per lane were subjected to 10% SDS–PAGE, transferred to PVDF membrane (Immobilon-P, Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), 20% methanol at 250 mA for 1.5 h, and the membrane was then blocked with PBS, 5% milk, 0.1% Tween 20. Next, the membrane was then probed sequentially with 1:3000 dilutions of the primary antibodies anti-GFP or anti-FLAG or anti-actin and goat anti-rabbit IgG-horseradish peroxidase diluted in PBS, 5% milk, 0.1% Tween 20. Blots were developed using the Clarity ECL Western blotting detection reagents (Bio-Rad), and images were acquired with the Tanon 4200 Chemiluminescent Imaging System (Tanon).

RNA preparation and quantitative RT-PCR

The mycelia were cultured for 18 h in liquid media and were then ground to a fine powder in liquid nitrogen. Total RNA was isolated using Trizol (Invitrogen, 15596–025) following the manufacturer’s instructions. 100 mg of mycelia per sample was used as the starting material for the determination of total RNA. The reverse transcription polymerase chain reaction (RT-PCR) was carried out using HiScript Q RT SuperMix (Vazyme, R123-01), and then cDNA was used for the real-time analysis. For real-time reverse transcription quantitative PCR (RT-qPCR), independent assays were performed using SYBR Premix Ex Taq (TaKaRa, DRR041A) with three biological replicates, and expression levels normalized to the mRNA level of actin. The 2^-ΔCT method was used to determine the change in expression.