*2.4. Manganese*

Manganese (Mn) is a transition metal and also an essential micronutrient in fungi. In agriculture, Mn compounds reduce mycelial growth of fungal pathogens [252,253]. In other pathogenic fungi, Mn2+ is required for virulence [254]. Some lignocellulose degrading enzymes also require Mn2+, such as manganese-dependent peroxidase, which white-rot fungi express during lignocellulose degradation, integral to nutrient uptake [255,256]. Many fungal species rely on Mn2+ and homeostatic mechanisms must exist to ensure proliferation.

#### 2.4.1. Manganese Transport and Homeostasis

Within *S. cerevisiae*, Mn2+ transporters Smf1 and Smf2 (part of the Nramp metal transporter family) and phosphate transporter Pho84, have a diverging consensus on their roles in Mn2+ homeostasis. In the case of Smf1, it was initially determined to be a highaffinity plasma membrane transporter, which acquired extracellular Mn2+ in Mn2+ deficient environments [65,66]. Smf2 is localized in golgi-like vesicles and shares approximately 50% identity with Smf1 (at the amino acid level), but does not share functionality and is a low-affinity Mn2+ transporter [77,257]. Once inside the cell, the fate of Mn2+ is as a cofactor for proteins such as Sod2 [126,258]. Sod2 is a mitochondrial manganese superoxide dismutase that receives Mn2+ via Mtm1 for activation [69,126,258,259]. In *smf2*∆ mutants, the Sod2 primary protein structure accumulates in the mitochondria; however, they were mostly inactive due to inadequate Mn2+ transfer to the mitochondria, indicating that Smf2 is a requirement for *S. cerevisiae* Sod2 activity [126,258]. Smf1 and Smf2, unlike many other metal ion transporters discussed in this review, are not regulated at the transcriptional level, rather post-translationally by protein turnover and localization, which is directly related to Mn2+ availability [260]. When Mn2+ concentrations are stable or in excess (~100 nmol/(1 × 10<sup>9</sup> cells)), Smf1 and Smf2 are ubiquitinated via Rsp5 (a NEDD4 family E3 ubiquitin ligase) with the aid of Bsd2 and transferrin receptor-like proteins (Tre1 and Tre2) [260–262]. Smf1 and Smf2 are then trafficked to multivesicular bodies, which deliver the proteins to the vacuole for degradation [260,263,264]. This mechanism of action is supported by reports that *tre1*∆, *tre2*∆, and *bsd2*∆ mutants resulted in the accumulation of Smf1 and Smf2 [260–262]. Conversely, when Mn2+ starvation occurs, Bsd2 is depleted, Smf1 is localized to the cell surface, Smf2 is localized to intracellular vesicles, and Smf1 and Smf2 resume their Mn2+ uptake functions [257,260,261].

The final transport system discussed here is the phosphate transporter Pho84. It was initially characterized in *S. cerevisiae* as a high-affinity, six-domain, transmembrane, inorganic phosphate transporter [265]. However, Pho84 is now also known as a lowaffinity Mn2+ transporter, along with other metals such as cobalt, zinc, and copper [67]. Through *pho84*∆ mutants, it was shown that Mn2+ uptake was the most commonly affected (in relation to the other metals) when *PHO84* was removed, further proving its Mn2+ transporter role [67]. *PHO84* transcription is regulated by transcription factor Pho4, which inhibits Pho84 activity when it is phosphorylated in the presence of excess phosphate; Pho4 resumes transcription when phosphate levels are low [265,266].

Once Mn2+ is inside the cell, there are an array of destinations. Pmr1 (high-affinity Ca2+/Mn2+ P-type ATPase) and Gdt1 (calcium/manganese transporter) both transport cytosolic Mn2+ to the Golgi lumen, where Mn2+ serves as a cofactor for mannosyl-transferases, such as Mnn1, Mnn2, Mnn5, and Mnn9, which glycosylate proteins in the secretory pathway [70–72,267–271]. This type of protein modification provides protein stability by preventing degradation, protecting against oxidative damage, and increasing thermodynamic equilibrium [272]. Concerning the ER, P-type ATPase Spf1 transports Mn2+ to the ER lumen; this is supported by a study showing that *spf1*∆ mutants had decreased luminal Mn2+; its overexpression had the opposite effect [73]. This same study also stated that Mn2+ depletion observed in *spf1*∆ mutants negatively impacted luminal Mn2+ dependent processes. On the contrary, it positively impacted Mn2+ associated cytosolic processes, indicating that Spf1 is integral to *S. cerevisiae* manganese ER and cytosolic homeostasis [73].

Mn2+ accumulation can have severe consequences on cellular health, and systems must be in place to prevent subsequent events. We will discuss two defense mechanisms in *S. cerevisiae*, Mn2+ trafficking to vacuoles for storage and degradation and Mn2+ export. Pmr1, previously characterized as an Mn2+ Golgi lumen transporter, also serves as a detoxifier. Presented with toxic Mn2+ levels, Mn2+ is still transported to the Golgi lumen from the cytosol, but excess ions are delivered to secretory pathway vesicles, which ultimately exit the cell, completely removing toxic Mn2+ (Figure 3) [77,273]. The *HIP1* gene product also expresses export activity. Hip1 was initially characterized as a high-affinity, plasma membrane histidine permease, but has since been shown to play a role in Mn2+ resistance [78,274]. Farcasanu et al. investigated *S. cerevisiae* mutants having defects in Mn2+transport and found that a mutation in the *HIP1* gene was responsible [78]. This mutation, originally a single base deletion, introduced a cascade of mutations that led to the protein Hip1-272 (272 amino acids long). Subsequent experiments showed that *hip1-272* mutants had significantly less cytosolic Mn2+ accumulation, increased Mn2+ efflux, and increased resistance than null mutants and wild type strains [78]. Further studies into the *hip1-272* mutant could elucidate the exact mechanisms of action of Mn2+ transport, determining how ions are trafficked to Hip1-272 and expelled. The second defense mechanism against Mn2+ toxicity in *S. cerevisiae* was Mn2+ trafficking to vacuoles through Ccc1 and Ypk9. Ccc1 (and possibly Cos16) is localized in the vacuolar membrane and is responsible for trafficking cytosolic Mn2+ to vacuoles; *CCC1* overexpression results in reduced Mn2+ toxicity, lower concentrations of cytosolic Mn2+, and increased vacuolar concentrations (Figure 3) [64,75,77]. Ypk9 is also localized in the vacuolar membrane and shuttles Mn2+ to the vacuole. Gitler et al. and Schmidt et al. both demonstrated that *ypk9*∆ mutants expressed Mn2+ hypersensitivity when compared to wild type strains, further affirming Ypk9 involvement in Mn2+ homeostasis [74,76].

**Figure 3.** Mn2+ uptake and detoxification systems in *S. cerevisiae*.

Δ Δ Manganese homeostasis has not been well characterized in higher fungi, but *Phanerochaete chrysosporium* has received some attention. *P. chrysosporium* is a white-rot fungus that produces lignin-degrading enzymes, which have been useful in the biodegradation of various plant biomass and an array of organo-pollutants [275–277]. Manganese peroxidase is a common lignin depolymerizing peroxidase utilized by white-rot Basidiomycetes [278,279]. It acts in combination with other enzymes to convert various biomass to useful bio-products of commerce and agricultural operations [255,280–283]. Homologs of the *S. cerevisiae* Pho84 and Smf1/2 proteins have been found in *P. chrysosporium*, PcPho84 and PcSmfs, respectively. PcPho84 is a plasma membrane protein involved in Mn2+ uptake, having a similar function to its *S. cerevisiae* homolog [68]. Smf1/2 are predicted to have similar functions in *P. chrysosporium* to their *S. cerevisiae* homologs [68]. Intracellular Mn2+ transport has also been investigated. Yeast homolog PcAtx2, localized in the Golgi membrane, was shown to function as an antioxidant through *sod1*∆ mutants [68]. When grown on 600 µM paraquat (inducer of oxidative stress), *sod1*∆ mutants experienced almost no growth; however, in mutants expressing *PcATX2*, growth was restored, indicating that PcAtx2 exhibits similar antioxidant functionality as Sod1 [68]. In the case of mitochondrial transport, *S. cerevisiae* Mtm1 traffics Mn2+ to the mitochondria for Sod1 activation; however, the function of the *P. chrysosporium* homolog, PcMtm1 (localized in the mitochondrial membrane), has yet to be identified, but predicted to have a similar antioxidant activity [68]. PcMnt and PcCcc1 engage in Mn2+ storage and export in *P. chrysosporium*, respectively. In *Phanerochaete sordida*, PsMnt was found to be a homolog of yeast Smf2 and plays a role in Mn2+ uptake, suggesting that it could have dual functionality, but this is still unknown [77,284]. Limited information exists on Mn2+ homeostasis in other fungi; however, due to the impact of Mn2+ on lignin-degrading enzymes in wood-rotting fungi, more studies should be conducted. Overall, Mn2+ homeostasis is critical to cellular functioning to prevent toxic Mn2+ accumulation, detoxify cells of free radicals, and provide white-rot fungi with their capacity to degrade lignin. In the absence of such mechanisms, toxicity can impede proper functioning and cause cellular damage.
