Molecular Characteristics of the Conserved Aspergillus nidulans Transcription Factor Mac1 and Its Functions in Response to Copper Starvation

Copper is an essential cofactor of enzymes during a variety of biochemical processes. Therefore, Cu acquisition plays critical roles in cell survival and proliferation, especially during Cu starvation. Knowledge of the key motif(s) by which the low-Cu-responsive transcription factor Mac1 senses Cu is important for understanding how Cu uptake is controlled. Findings in this study demonstrated that the Cu fist motif, but not Cys-rich motifs, is essential for Mac1-mediated Cu uptake in Aspergillus. In addition, Cu transporters CtrA2 and CtrC are both required for Mac1-mediated Cu uptake during Cu starvation in A. nidulans, indicating that species-specific machinery exists for Cu acquisition in Aspergillus.


RESULTS
Functional conservation of low-Cu-responsive transcription factor Mac1 in selected fungal species. All selected Mac1 homologs contain an N-terminal Cu fist domain and C-terminal Cys-rich motifs, each containing five cysteines and one histidine residue (Fig. 1A). This similarity in protein architecture led us to hypothesize that A. fumigatus Mac1 (AfMac1) homologs could functionally replace each other. In order to test this hypothesis, we performed homolog replacement assays in the A. fumigatus ⌬Afmac1 background strain. The complementation strains were generated by individually introducing the A. nidulans mac1 (Anmac1), S. cerevisiae mac1 (Scmac1), and Schizosaccharomyces pombe cuf1 (Spcuf1) genes, under the control of the Afmac1 native promoter, into the ⌬Afmac1 mutant. As shown in Fig. 1B, introduction of ⌬Afmac1 AnMac1 (CZD01) restored the ⌬Afmac1 strain to the wild-type phenotype under conditions of Cu starvation stress, suggesting that AnMac1 can functionally complement AfMac1. In contrast, strains complemented with Scmac1 and Spcuf1 (strains ⌬Afmac1 Scmac1 and ⌬Afmac1 Spcuf1 [CZD02 and CZD03]) still exhibited the ⌬Afmac1-like phenotype, with sparse conidia and shortened hyphae, under conditions of Cu starvation. Furthermore, semiquantitative reverse transcription (RT)-PCR analysis demonstrated that the two yeast homolog replacement strains exhibited comparable expression levels of Scmac1 or Spcuf1 in the background of the Afmac1 gene deletion strain, suggesting that Scmac1 and Spcuf1 were normally expressed in the Aspergillus system at the mRNA level (see Fig. S1 in the supplemental material). The data indicate that, unlike the yeast Mac1 homologs, A. nidulans Mac1 may have a conserved function similar to that of the corresponding A. fumigatus homolog under low-Cu conditions. We next investigated the function of the conserved Cys residues that are part of the homologous structures in both the Cu fist domain and the Cys-rich motifs in Mac1 homologs (Fig. 1A). A mutant AnMac1 variant was created by modifying the first Cys residue of AnMac1 at position 12 to Ser and then introduced it into the ⌬Afmac1 mutant. The resulting strain was referred to as CZD04 [⌬Afmac1 AnMac1(C12S) ]. In contrast to the wild-type strain, the Cys-mutated AnMac1 strain exhibited ⌬Afmac1-like defects with shortened hyphae and reduced production of conidia, as observed microscopically, suggesting that the Cys residue at the position 12 is required for the normal function of AnMac1 during Cu starvation (Fig. 1B). This further underlines the potential importance of Cys residues within AnMac1.
AnMac1 is responsible for hyphal growth and conidiophore development during Cu starvation. As shown in Fig. 1, AnMac1 was able to functionally substitute AfMac1 for colony growth under conditions of Cu starvation. To further explore the function of Mac1 in Cu starvation in A. nidulans, we knocked out the ANIA_00658 gene in A. nidulans parental strain TN02A7 to generate a ⌬Anmac1 mutant. This was achieved by replacing the gene's open reading frame (ORF) with the A. fumigatus pyrG marker, generating strain CZD05 (⌬Anmac1). Compared to the parental wild-type strain (A. nidulans WT [AnWT]), the ⌬Anmac1 mutant showed colonies with significantly reduced radial hyphal growth and fewer conidia under low-Cu conditions ( Fig. 2A). This growth phenotype was rescued by the Anmac1 gene, as demonstrated in Anmac1complemented strain CZD06 (Anmac1 c ; Fig. 2A). As expected, exogenous Cu supplementation resulted in significant rescue of the defective phenotypes in the ⌬Anmac1 mutant in a dose-dependent manner, indicating that Anmac1 regulates Cu uptake under low-Cu conditions. To further determine the cellular function of AnMac1, we generated a green fluorescent protein (GFP)-labeled AnMac1 strain (CZD07), expressing an AnMac1 C-terminal GFP-tagged fusion protein driven by an AngpdA constitutive promoter, in the ⌬Anmac1 mutant background. As expected, the AnMac1-GFP strain fully complemented the defects associated with loss of AnMac1 (Fig. S2), suggesting that the AnMac1-GFP fusion protein was functional. Importantly, cells expressing AnMac1-GFP showed strong nuclear localization signals, as demonstrated by DAPI (4=,6-diamidino-2-phenylindole)-stained nuclear distribution (Fig. 2B). The data suggest that AnMac1 is a nuclear localized transcription factor.
Comparison of the colony morphologies of the WT and ⌬Anmac1 mutant strains revealed that the WT strain produced blue-green spores, unlike the ⌬Anmac1 mutant, which produced smaller, white conidia. Using a previously described sandwich cover- slip protocol (31), we determined that vegetative mycelia of the WT strain developed into complete conidiophores with visible phialides connected by chains of numerous conidia, whereas the ⌬Anmac1 mutant produced severely abnormal structures of conidiophores with very few conidia. A phenotype similar to that of the ⌬Anmac1 mutant was observed in the AnMac1 C12S strain (Fig. 2C). These results imply that AnMac1-mediated Cu regulation is involved in the development of conidiophores and conidia.
The Cu fist motif, but not the C-terminal Cys-rich motif, is required in Mac1 for its function in Cu starvation. To further assess the roles of the conserved Cys residue within the Cu fist domain, Cys residues at positions 12, 15, and 24 were individually mutated to Ser in the Cu fist domain of AnMac1 and were introduced into the ⌬Anmac1 background strain under the control of the Anmac1 native promoter (Fig. 3A). The resulting AnMac1 mutant strains were referred to as CZD08 (AnMac1 C12S ), CZD09 (AnMac1 C15S ), and CZD10 (AnMac1 C24S ). In contrast to the WT strain, the three Cys mutants exhibited severe growth defects, with smaller colonies and fewer conidia, suggesting that these Cys residues are required for the normal function of AnMac1 under Cu starvation conditions (Fig. 3B). Similarly, by generating individual site-directed mutations, we found that conserved RGHR and GRP residues within the Cu fist domain are important for the AnMac1-mediated response to low-Cu conditions, as shown in strains CZD11 (AnMac1 RGHR to AAAA ) and CZD12 (AnMac1 GRP to AAA ) (Fig. 3B). These data suggest that the Cu fist motif harboring the conserved Cys, RGHR, and GRP residues is essential for Mac1 function in response to Cu starvation. To ascertain the function of the conserved REP-I and REP-II motifs within the C terminus of AnMac1 ( Fig. 1A and Fig. 3A), all of the Cys and His residues were mutated in each independent REP element. Unexpectedly, mutants AnMac1 REP-I , AnMac1 REP-II , and AnMac1 REP-IϩII (CZD13, CZD14, and CZD15) all showed normal colony phenotypes comparable to those shown by the WT strain under Cu starvation conditions (Fig. 3C). This finding suggests that, under Cu starvation conditions, the C-terminal Cys-rich motifs are dispensable for AnMac1 function. Using the same strategy, we also demonstrated that the Cys-rich motifs are not required for AfMac1 function in Cu starvation in A. fumigatus, as shown by the AfMac1 REP-I and AfMac1 REP-II mutants (CZD16 and CZD17) (Fig. 3D). In summary, we conclude that N-terminal Cys residues play an important role in Mac1 function in the response of Aspergillus to low-Cu conditions, while the C-terminal Cys-rich motifs in AnMac1 are dispensable.
Cu transporters CtrA2 and CtrC are both required for Mac1-mediated Cu uptake during Cu starvation in A. nidulans. Previously, we demonstrated that Mac1-mediated Cu uptake depends on transporters CtrA2 and CtrC in A. fumigatus (27). Using BLAST searches, we identified A. fumigatus ctrA2 and ctrC homologs in A. nidulans as ANIA_03209 and ANIA_03813, here referred as AnctrA2 and AnctrC. Furthermore, a predicted structure analysis showed that both AnCtrA2 and AnCtrC contain multiple transmembrane domains and Met-X 1-5 -Met motifs (Fig. S3A), which are typical signatures of the Cu transporters (32,33). To elucidate the molecular mechanisms underlying the Mac1-mediated low-Cu response in A. nidulans, we examined the mRNA levels of AnctrA2 and AnctrC in AnMac1 mutation strains. As shown in Fig. 4A and B, AnctrA2 and AnctrC were significantly downregulated in the ⌬Anmac1 and AnMac1 C12S mutants, indicating that AnctrA2 and AnctrC were transcriptionally dependent on the presence of AnMac1. On the basis of real-time reverse transcription-quantitative PCR (RT-qPCR) data, we overexpressed AnctrA2 and AnctrC driven by the AngpdA constitutive promoter in the ⌬Anmac1 mutant. These strains were referred to as CZD18 and CZD19 (mutants ⌬Anmac1 OE::AnctrA2 and ⌬Anmac1 OE::AnctrC ), respectively. Furthermore, we found that the expression levels of AnctrA2 and AnctrC were significantly elevated in overexpression strains ⌬Anmac1 OE::AnctrA2 and ⌬Anmac1 OE::AnctrC , respectively, compared to the level seen with the ⌬Anmac1 strain (Fig. 4C). These data suggest that AnctrA2 and AnctrC were truly overexpressed but could not fully rescue defects induced by loss of AnmacA. As unpredicted, the strain overexpressing either AnctrA2 or AnctrC still displayed a defective colony phenotype upon Cu starvation (0 M addition). Partial rescue of colony growth was achieved upon addition of Cu at a concentration of 1 or 10 M (Fig. 4D). The data indicate that overexpression of AnCtrA2 or AnCtrC alone was unable to rescue the phenotype induced by the deletion of AnMac1 and that synergistic functioning of AnCtrA2 and AnCtrC seems to be required in the AnMac1mediated Cu starvation response of the cell.
To further illuminate the function of AnctrA2 and AnctrC in response to low-Cu environments, we constructed single and double deletions of AnctrA2 and AnctrC. As seen in Fig. 4E, single deletion mutants ⌬AnctrA2 and ⌬AnctrC (CZD20 and CZD21) exhibited WT-like colony morphologies, whereas the double deletion mutant, ⌬AnctrA2 ⌬AnctrC (CZD22), showed severe defects in low-Cu environments, especially under Cu starvation conditions. Notably, addition of exogenous Cu to the media (at 1 and 10 M Cu) rescued the defective phenotypes of the ⌬AnctrA2 ⌬AnctrC double deletion strain. In comparison, 1 M Cu was unable to rescue the colony defects in a ⌬Anmac1 mutant, whereas phenotype rescue was seen at 10 M and 20 M. Thus, the data suggest that higher concentrations of Cu addition can bypass the requirement of the AnMac1-Ctr pathway and that Mac1 plays a more important role in Cu uptake than regulatory Cu transporters CtrA2 and CtrC in A. nidulans.

DISCUSSION
Copper acquisition is important for cell survival and proliferation, especially during periods of Cu starvation. The low-Cu-responsive transcription factor Mac1 and its regulated Cu transporters have been reported in A. fumigatus. However, the key motif(s) by which Mac1 senses Cu has not been defined and is important for understanding how Cu uptake is controlled in Aspergillus. In this study, site-directedmutagenesis experiments, combined with homolog complementation assays, demonstrated that the Cu fist motif is essential, but not sufficient, for AnMac1-mediated Cu uptake during Cu starvation. Our findings may have broad implications for the structurally conserved Mac1 homologs in Aspergillus. In addition, unlike their counterparts in A. fumigatus (27), overexpression of the transporters AnCtrA2 and AnCtrC cannot functionally compensate for the loss of AnMac1 under the low-Cu culture conditions. Further, our data suggest that AnCtrC is responsible for low-Cu response only in the absence of AnCtrA2, indicating that the conserved Mac1-mediated Cu uptake in A. fumigatus and A. nidulans also possesses species-specific machinery.
Our data further demonstrated that A. nidulans Mac1 could functionally crosscomplement A. fumigatus Mac1 deletion in response to low-Cu conditions, indicating that the Cu uptake mechanism may be conserved in Aspergillus. However, there was a significant difference in the threshold value of Cu required for the phenotype rescue. Addition of 20 M Cu sulfate in minimal media resulted in an almost complete restoration of the WT phenotype in the ⌬Anmac1 mutant, whereas the ⌬Afmac1 mutant required more than 100 M Cu to be rescued. This finding suggests that, in addition to Mac1, A. nidulans may have other high-affinity Cu-uptake systems to bypass the requirement of AnMac1, while A. fumigatus may not (27). Importantly, overexpression of either Cu transporter AnCtrA2 or Cu transporter AnCtrC in A. nidulans failed to completely restore the ΔAnmac1 mutant to the WT phenotype during Cu starvation. This implies that, in contrast to A. fumigatus (27), in the absence of AnMac1, Cu transporters AnCtrA2 and AnCtrC may need to function together. Another possibility is that there might be some unknown AnMac1-mediated targets that also play a role in the Cu uptake process in A. nidulans. In addition, we demonstrated that the CtrC Cu transporter in A. fumigatus, but not that in A. nidulans, is responsible for the Cu starvation response. Furthermore, the alignment analysis showed that the level of amino acid sequence identity of AnCtrC and AfCtrC was 45.3%, while AnCtrA2 showed only 11% sequence identity with AfCtrA2 but 34.5% sequence identity with AnCtrC (see Fig. S3B in the supplemental material). The result suggests that the CtrC Cu transporter but not CtrA2 possess high conservation between A. nidulans and A. fumigatus, implying that CtrA2 may have different functions for Cu uptake in A. nidulans and A. fumigatus. Collectively, these results suggest that the conserved Mac1-mediated Cu uptake in A. fumigatus and that in A. nidulans also have their own unique styles.
In S. cerevisiae, both Cu fist structures and Cys-rich motifs are involved in Cudependent DNA binding, indicating that they are potentially important for Mac1 function in Aspergillus. Site-directed mutagenesis demonstrated that the conserved Cu fist motif is responsible for the AnMac1-mediated Cu starvation response. Furthermore, data from site-directed mutagenesis and homolog replacement experiments led us to conclude that the conserved Cu fist domain is essential, but not sufficient, for the AfMac1-mediated functional response to low-Cu conditions. This indicated that the conserved Cu fist motif requires cooperation with nonconserved regions to accomplish Mac1-mediated low-Cu response in Aspergillus. Interestingly, the Cu fist domain is conserved among the members of the family of Cu-responsive transcription factors (27,34). We have previously demonstrated that the Cu fist motif is required for the function of the high-Cu-sensing transcription factor AceA in Cu detoxification in A. fumigatus (34). Therefore, both high-Cu-sensing and low-Cu-sensing transcription factors may utilize a common mechanism to bind DNA in A. nidulans and A. fumigatus. However, in S. cerevisiae, the Cu detoxification transcription factor Ace1 utilizes N-terminal Cys-X 1-2 -Cys motifs to bind Cu ϩ ions in a polycopper cluster, in contrast to the C-terminal Cys-rich motifs in REP-I and REP-II in Mac1 (13,16,20,21). This difference implies that high-Cu-sensing and low-Cu-sensing transcription factors may have distinct Cu-binding domains in yeasts. Notably, we found that the REP-I and REP-II motifs are dispensable for Mac1 function in Cu starvation in both A. nidulans and A. fumigatus, suggesting that they possess Cu-ion binding characteristics that are distinct from those possessed by yeasts. However, we still cannot exclude the possibility that REP-I and REP-II motifs may play a role in Mac1 function in high-Cu-induced degradation of Cu transporters analogous to the role seen in S. cerevisiae (21).

MATERIALS AND METHODS
Strains, oligonucleotides, media, and transformation. Lists of all the Aspergillus strains and oligonucleotides used in this study are provided in Table S1 and Table S2, respectively. The TN02A7 deletion strain of a gene required for nonhomologous end joining in double-strand break repair (35,36) was used to generate the ⌬Anmac1, ⌬AnctrA2, and ⌬AnctrC mutant strains. All A. fumigatus strains were grown on minimal MM solid medium (1% glucose, 1 ml liter Ϫ1 trace elements, 50 ml liter Ϫ1 20ϫ salt [pH 6.5], and 2% agar), while all of the A. nidulans strains were grown on minimal MMPDR solid medium (2% glucose, 1 ml liter Ϫ1 trace elements, 50 ml liter Ϫ1 20ϫ salt, 0.5 mg liter Ϫ1 pyridoxine, 2.5 mg liter Ϫ1 riboflavin, 2% agar, pH 6.5) (27,37,38). The agar was omitted for liquid medium. Notably, the content of CuSO 4 was removed from the trace elements as previously described (39), with a few modifications, such that Cu was absent in both the MM and the MMPDR media used in this study. Transformation was performed following published protocols (40,41).
Construction of strains for AnMac1 homolog replacement experiments. To construct an Anmac1 open reading frame (ORF) driven by the Afmac1 native promoter, fragments containing regions flanking the 5= and 3= Afmac1 ORF were amplified from A. fumigatus A1160 genomic DNA with primer pairs Afmac1(p)-F/Afmac1(p)-An-R and Afmac1(t)-F/Afmac1(t)-R. The coding sequence of AnMac1 ORF was amplified with primer pair Anmac1(ORF)-S/Anmac1(ORF)-A. The resulting three PCR products were Cu and shaken on a rotary shaker at 220 rpm and 37°C for 24 h. All data were obtained on the basis of results from three independent experiments.