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

Copper (Cu) is an essential trace element in all organisms, and Cu acquisition during periods of starvation is important for cell survival and proliferation. Although the Cu starvation-responsive transcription factor Mac1 as well as its targeted Cu transporters have been identified in Aspergillus fumigatus, the molecular mechanisms of Mac1-mediated Cu acquisition have not yet been investigated in Aspergillus. We demonstrated that Mac1 and its regulated Cu transporters are required for growth and conidiophore development during Cu starvation in Aspergillus nidulans. Moreover, A. nidulans Mac1 (AnMac1) showed highly functional conservation with the A. fumigatus homolog but not with homologs in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Molecular characterization of Mac1 in A. nidulans demonstrated that the “Cu fist” motif (i.e., residues 1 through 40) harboring Cys, RGHR, and GRP residues is required for the Mac1-mediated low-Cu response but not the Cys-rich motifs REP-I and REP-II. Notably, overexpression of either the CtrA2 Cu transporter or the CtrC Cu transporter individually was unable to functionally rescue the defects in the AnMac1 deletion strain, implying that Cu uptake might require both CtrA2 and CtrC during Cu starvation, which is different from results seen with A. fumigatus. Findings in this study further suggest that the conserved Mac1-mediated Cu uptake machinery in A. fumigatus and A. nidulans is also species specific. IMPORTANCE 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.