*2.2. Copper*

Copper is also a transition metal and presents itself in oxidation states copper(I), Cu<sup>+</sup> , and copper(II), Cu2+ [32,48]. It is essential to agriculture and human medicine where it can serve as a fungicidal or fungistatic agent, or be the determining factor for virulence [174,175]. Some fungal pathogens heavily rely on copper exporters to prevent host-enacted copper toxicity or import machinery to maintain virulence. In both clinical and agricultural settings, fungal exposure to excess copper can result in ionic imbalance. Therefore, homeostatic mechanisms to maintain healthy intracellular copper levels are critical.

#### 2.2.1. Copper Transport and Homeostasis

Generally, copper cannot permeate the plasma membrane and requires membrane transporters for uptake [32,48]. Before internalization, copper must exist as Cu<sup>+</sup> (cuprous oxide); however, in the environment, it often exists as Cu2+ (cupric oxide) and must undergo reduction. In *S. cerevisiae*, cupric reductase Fre1, transcribed by Mac1, reduces Cu2+ to Cu<sup>+</sup> , making it readily available for uptake via high–affinity membrane transporters of the copper transporter (Ctr) protein family, Ctr1 and Ctr3 or low-affinity copper transporter Fet4 (Figure 2) [30–33,176]. Transcription of *CTR1* and *CTR3* is also regulated by transcription factor Mac1, which regulates transcription based on copper availability; copper depletion results in the upregulation of *CTR1*/*3,* and copper repletion results in downregulation [30,177].

After uptake, Cu<sup>+</sup> serves as enzymatic cofactors. Apoproteins within the secretory pathway require copper for proper functioning, such as the multicopper oxidase Fet3, which is necessary for ferrous iron, Fe(II), uptake, and oxidation [53,178–180]. *FET3* is regulated by transcription factor Aft1 (activator of ferrous transport) in iron-deficient conditions and its gene product contains four Cu<sup>+</sup> binding sites where copper serves as a cofactor for enzyme activation [53,178,181]. Unmetalated Fet3 reduces cell growth in iron-limiting conditions, demonstrating the importance of copper transport [44,182].

Another enzyme dependent on copper is the cytoplasmic Cu/Zn superoxide dismutase (Sod1). This is an antioxidant for superoxide anions (O<sup>2</sup> •−) [183,184]. O<sup>2</sup> •− are ROS that cause cellular damage and toxicity and must be effectively dismutated to prevent stress; therefore, delivery of copper to Sod1 is critical [45,185,186]. In *S. cerevisiae*, the cytosolic copper chaperone Lys7 acquires Cu<sup>+</sup> and delivers it to Sod1, with high specificity [45]. Once Sod1 is metalated, it is then able to catalyze the dismutation reaction that results in O<sup>2</sup> •− being successfully detoxified to hydrogen peroxide (H2O2) and molecular

oxygen (O2); H2O<sup>2</sup> is now readily available for further detoxification to water via catalyst Cct1 [183,187,188]. Cu<sup>+</sup> transport to MTs Cup1 (also known as Cup1-1 and Cup1-2) and Crs5 is also integral to cellular detoxification [18,189]. Both MTs are regulated by transcription factor Ace1 (also known as Cup2), which activates the transcription of *CUP1* and *CRS5* at elevated copper concentrations [167,189]. Cup1 and Crs5 contain 8 and 11-12 Cu<sup>+</sup> binding sites, respectively, and are responsible for buffering cytosolic copper to maintain safe intracellular copper concentrations [189–191]. Though Crs5 has a greater copper binding capacity, it plays a much smaller role in detoxification due to its promoter region, which only has one recognition sequence, compared to four in *CUP1* [189–191].

*S. pombe* follows a pattern of copper transport similar to *S. cerevisiae.* Extracellular Cu2+ is reduced to Cu<sup>+</sup> by cell surface reductases before uptake [34,36]. Cu<sup>+</sup> can then be transported across the cell membrane, depending on the current cell cycle [34–36]. During mitosis, an integral membrane complex composed of proteins Ctr4 and Ctr5 are responsible for Cu<sup>+</sup> uptake, and during meiosis, Mfc1 (localized in the forespore membrane) is responsible [34–36]. Expression of ctr4<sup>+</sup> and ctr5<sup>+</sup> is regulated by transcription factor Cuf1, and expression of mfc1<sup>+</sup> is regulated by transcription factor Mca1, both of which are activated or deactivated by the absence or presence of sufficient copper levels, respectively [34,36]. Once inside the cell, copper chaperones such as Cox17, Pccs, and Atx1 transport Cu<sup>+</sup> to respective organelles [46]. Pccs is a four domain, cytosolic chaperone. The first three domains are responsible for transporting Cu<sup>+</sup> to unmetalated Sod1 in a copper-limited environment, activating Sod1 [46]. In high copper environments, the fourth domain acts as a copper buffering system, sequestering Cu<sup>+</sup> to prevent toxic cytosolic levels [46]. Atx1 in *S. pombe* plays a similar role to Atx1 in *S. cerevisiae*. In *S. pombe*, Atx1 is also located in the cytosol and responsible for carrying Cu<sup>+</sup> to Ccc2 [34,42]. Peter et al. and Beaudoin et al. described how Atx1 was also used for copper transport to copper amine oxidases (CAOs), a group of catalysts not present in *S. cerevisiae* [34,42]. Atx1 shuttles Cu<sup>+</sup> to an active site on the CAO, where copper (and another required cofactor, 2, 4, 5-trihydroxyphenylalanine quinone) activates it [34,42,192]. *S. pombe*'*s* Cox17 is an orthologue to *S. cerevisiae* Cox17, sharing 38% identity and is located in the mitochondrial intermembrane space [42,48]. Once Cox17 acquires Cu<sup>+</sup> it is delivered to Sco1, Sco2, and Cox11 for copper loading to cytochrome c oxidase subunits [42,47,48].

Filamentous fungi are also important in assessing copper homeostasis, as these organisms depend on copper for growth and virulence in pathogenic species. In the pathogenic Ascomycete *Aspergillus fumigatus*, studies have shown similarities to *S. cerevisiae* and *S. pombe* in copper uptake. Cu2+ must also be reduced before uptake, however, there is some ambiguity regarding the reductases responsible [39]. This reductase has been referred to as unknown ferric reductase ("Fre?"), a general Fre reductase, and metallo-reductase Afu8g01310 (homolog of *S. cerevisiae FRE* or *FRE3*) [39,193,194]. After reduction, CtrA2 and CtrC (both homologs of *S. cerevisiae* Ctr1) transport Cu<sup>+</sup> into the cytosol and serve as enzymatic cofactors [37,39]. CtrA2 and CtrC are regulated by transcription factor MacA (also referred to as AfMac1) which senses low copper concentrations and activates CtrA2 and CtrC [39,49,195,196]. Conversely, in high copper concentrations, transcription factor AceA activates P-type ATPase CrpA as a defense mechanism for copper export and is responsible for extended life and virulence [39,49,195,196].

Limited knowledge exists on copper homeostasis in Basidiomycetes. Studies in two Basidiomycetes, the brow-rot fungus *Fibroporia radiculosa* and the edible white-rot fungus *Pleurotus ostreatus*, reported some details. In *F. radiculosa*, only the regulation of intracellular Cu<sup>+</sup> concentration has been unveiled, by three, unnamed copper ATPases and one gene of unknown function, CutC, [197]. In *P. ostreatus*, membrane protein Ctr1 is involved in copper uptake and shares homology with the low-affinity copper transporter PaCtr2 of the Ascomycete *Podospora anserine* (20%) and the high-affinity *S. cerevisiae* copper transporter, Ctr1 (20%) [38]. This review shows that copper homeostasis is well-studied in *S. cerevisiae* and *S. pombe*; however, more research is needed in other Ascomycetes and Basidiomycetes.
