silencing altered Cu homeostasis as predicted, suggesting a conserved role. For example, knockdown (KD) of Ctr1B reduced Cu uptake and content, whereas downregulation of DmATP7 induced Cu accumulation due to a deficient efflux. As a result, the systemic Cu homeostasis is very similar between humans and Drosophila melanogaster (Southon et al., 2013a). **Figure 1** and **Table 1** summarize the key fly proteins involved in Cu metabolism. One of the main handicaps of the studies about Cu biology is the lack of proper and direct ways to measure Cu and to monitor differences either in the amount or in the distribution of the metal. Richard Burke's lab has very elegantly and nicely showed that synchrotron x-ray fluorescence microscopy is able to detect Cu alterations in a small organism such as Drosophila even in some tissues including the fly brain (Lye et al., 2011).

#### Cu Uptake

Copper transporter 1 (CTR1) is the main responsible of cellular import of Cu. CTR1 belongs to the SLC31 family of transporters and forms a trimeric channel to allow Cu uptake (Nevitt et al., 2012). The fly genome contains three different orthologs (**Figure 1**): Ctr1A, Ctr1B, Ctr1C (Zhou et al., 2003). Ctr1A is highly expressed throughout development, whereas Ctr1B seems to be only relevant in late embryo and larvae with very strong expression in enterocytes (in order to uptake Cu from food). Ctr1C is very lowly expressed in all developmental stages and the expression seems to be mainly focused in male gonads (Zhou et al., 2003; Hua et al., 2010). All three proteins seem to be mainly localized in the plasma membrane (Selvaraj et al., 2005; Turski and Thiele, 2007; Binks et al., 2010; Steiger et al., 2010). In agreement, they are able to stimulate Cu import into Drosophila S2 cells (Zhou et al., 2003) and also in vivo (Balamurugan et al., 2007; Hua et al., 2010). Ubiquitous or panneuronal overexpression of any of the three genes is developmentally lethal, whereas eye-specific overexpression triggered a rough eye phenotype accompanied by neuronal degeneration (Balamurugan et al., 2007; Hua et al., 2010; Lang et al., 2013). This retinal degeneration is clearly related to Cu availability, since reducing Cu content in retinal neurons by increasing efflux (Balamurugan et al., 2007), enhancing Cu chelation by Mtns (Egli et al., 2006a), or with bathocuproine disulfonate (BCS) (Balamurugan et al., 2007; Hua et al., 2010) can rescue the phenotype.

Ctr1A seems to be the most important of all fly copper importers, since it displays the broadest expression pattern and its complete loss-of-function triggers developmental arrest (Turski and Thiele, 2007; Hua et al., 2010). In agreement with a reduced Cu import, Ctr1A deficient larvae and adults show lower activity of Cu-requiring enzymes such as Cytochrome c Oxidases (Coxs), tyrosinase (Tyr) and peptidylglycinehydroxylating monooxygenase (Phm). This resulted in lower pigmentation of mouth hooks and body, and reduced amidation of neurotransmitters (Turski and Thiele, 2007; Binks et al., 2010). Importantly, Cu-supplementation in the food (Turski and Thiele, 2007; Hua et al., 2010) or genetic manipulation of intracellular copper levels (Binks et al., 2010) improved some of the defects. Furthermore, the human counterpart hCtr1 also improved loss of Ctr1A conditions, suggesting a strong functional equivalence between both proteins (Hua et al., 2010). Due to the lethality associated to the complete depletion of Ctr1A, tissue-specific experiments have also been performed. KD in the dorsal vessel generated a severe cardiomyopathy in the flies (Kim et al., 2010). This result demonstrates that cardiac defects present in several models of systemic Cu deficiency are a result of a tissuespecific effect. It is likely that, in a tissue highly dependent on mitochondria, strong reduction of Cox activity is underlying the cardiac defect. Ctr1A depletion in the nervous system showed that it is required for neuronal development (Binks et al., 2010). Interestingly, KD of Ctr1A in the protothoracic gland induced phenotypes reminiscent to a reduced Ras signaling. Accordingly, downregulation of Ctr1A was able to rescue the overactivation of Ras (Turski et al., 2012). Ctr1B is the only Cu importer identified as a direct target of dMTF-1 (Southon et al., 2004; Selvaraj et al., 2005). Ctr1B is upregulated by Cu deprivation (Zhou et al., 2003) in order to maintain the correct systemic Cu amount and, unexpectedly, also by toxic accumulation of mercury and cadmium (Balamurugan et al., 2009). Mutants are viable but show hypopigmentation due to defects in the activity of the Cucontaining enzyme Tyr. Since those mutants do not accumulate Cu, they fail to induce the expression of Mtns (Zhou et al., 2003; Balamurugan et al., 2007). This defect is really Cu specific since mutants successfully promoted Mtns expression upon feeding with cadmium (Balamurugan et al., 2007). However, under conditions of Cu scarcity, mutants are unable to complete development (Zhou et al., 2003). In agreement with the specific expression pattern in the fly gut, a panneuronal KD of Ctr1B does not affect cellular integrity (Binks et al., 2010). Ctr1C is the least studied and its expression seems to be restricted to the maturing spermatocytes and the mature sperm. Ctr1C mutants are viable but their fertility is severely compromised in a Cudependent manner (Steiger et al., 2010). Malvolio (Mvl), the Drosophila homolog of Divalent metal transporter-1 (DMT1) is mostly involved in the transport of dietary iron in the gut (Folwell et al., 2006) but also seems to influence Cu content in Drosophila S2 cells (increased when overexpressed and reduced when depleted) and in the fly gut (Southon et al., 2008). Mutant individuals are viable but sensitive to increased levels of Cu in the food. Since its function might be redundant with members of the Ctr1 family, it is logical that the loss-of-function does not cause a systemic Cu deficiency.

#### Cu Efflux

In mammals, the responsibility to secrete Cu from cells relies on two P-Type ATPases, ATP7A, and ATP7B. They both belong to a group of genes that hydrolyze ATP to pump substrates across membranes (Zlatic et al., 2015). The fly genome only encodes one ortholog of mammalian ATP7A and B, DmATP7 (**Figure 1**) which displays a high homology (>45%) to both ATPases (Southon et al., 2004). However, comparative genomic analysis of 12 Drosophila species shows that DmATP7 contains all the motifs existing in ATP7A required for localization and retention in the basolateral membrane, whereas ATP7B targeting motifs are not present (Southon et al., 2010). ATP7A has been described as a multitasking protein. It seems to be initially located in the transgolgi network (TGN), where it participates in the transfer of Cu into Cu-containing proteins and it is relocated to the plasma membrane upon increasing concentrations of Cu (Kaler, 2011), where it is involved in cellular efflux of Cu. Several functional reporter constructs (DmATP7-lacZ, EYFP-DmATP7, UAS-DmATP7FLAG, gDmATP7:GFP) have been used to study the endogenous expression pattern of DmATP7. DmATP7 is highly expressed at the basolateral membrane of the Cu cells of the midgut and in the central, peripheral and enteric nervous system in all developmental stages. It is also expressed in the larval mouthparts, developing tracheae, the gut and in the malpighian tubules but not in the optic lobes, salivary glands and fat body. In the adult nervous system the expression in peptidergic neurons is of special importance due to the role of Cu in the biosynthesis of amidated neurotransmitters (Norgate et al., 2006; Burke et al., 2008; Sellami et al., 2012; Mercer et al., 2017). In all cases, fusion proteins show clear localization at the plasma membrane. Importantly, DmATP7 presents a Cuinducible and dMTF-1 dependent expression (Burke et al., 2008; Mercer et al., 2017). It is known that the Cu-induced trafficking of mammalian ATP7A and ATP7B from the TGN toward the plasma membrane is critical for their role in Cu homeostasis (Kaler, 2011). Unexpectedly, this trafficking was not observed in cultured embryonic S2 cells, larval neuronal Bm3-c2 cells (Southon et al., 2010) or in vivo in larval midgut cells (Burke et al., 2008; Mercer et al., 2017) with any of the DmATP7 constructs. However, DmATP7 is able to translocate from the TGN to the plasma membrane when introduced in mammalian cells (Southon et al., 2010). Therefore, fly models still need to clarify whether such translocation is not required/necessary in flies or whether just current methodological limitations avoid its detection. In agreement with the expected role in Cu transport and export, coexpression of DmATP7 counteracted the phenotypes induced by the overexpression of the Cu importer (Ctr1B) in the Drosophila retina (Balamurugan et al., 2007). Panneural overexpression of DmATP7 induced an altered morphology of neuromuscular junctions (Comstra et al., 2017) followed by a partial developmental lethality and a high percentage of unexpanded wings in the surviving adults due to a deficient activity of the corresponding neurotransmitters (Hwang et al., 2014). Similarly, ubiquitous overexpression induced body hypopigmentation due to the impairment of the Cu-requiring enzyme Tyr (Norgate et al., 2006). Since all these effects are rescued by Cu-supplemented food, we can conclude that overexpression also results in Cu deficiency due to increased efflux (Norgate et al., 2006; Hwang et al., 2014).

#### Cu Chaperones

In addition to transporters of Cu into and out of the cells, there is a third group of proteins with critical roles in Cu homeostasis. They are the so called "Cu chaperones" since their main role is to deliver Cu into aproproteins of cuproenzymes (Palumaa, 2013). Drosophila orthologs (**Figure 1**) of the mammalian Cu chaperones ATOX1, CCS, COX17 and SCO1, and SCO2 are found to be expressed in Drosophila S2 cells (Southon et al., 2004). Atox1 is the fly homolog of the mammalian ATOX1, which delivers Cu to ATP7A and ATP7B. In agreement, loss of Atox1 function in the flies phenocopies most of the defects of DmATP7 mutant flies (Hua et al., 2011a). CCS is the chaperone that delivers Cu to SOD1 (Cu/Zn superoxide dismutase). Ccs mutant flies display low levels of SOD1 activity, but also reduced levels of SOD1 protein. This important evidence suggests that Ccs plays a role in the stability of SOD1 (Kirby et al., 2008). SCO proteins are the Cu chaperones in charge of delivering Cu to the mitochondrial cytochrome c oxidase (COX). The Drosophila genome only encodes a single ortholog named Scox that is expressed during the whole development. Correspondingly, null mutants show a severely reduced COX activity that triggers developmental arrest at L2 stage. Hypomorphic alleles affect locomotion and female fertility, in agreement with its high expression in the egg chambers (Porcelli et al., 2010). It is likely that mitochondrial dysfunction is underpinning the loss-of-scox defects. However, additional studies will be needed to find out the complete mitochondrial mechanism. Unfortunately, although homologs of mammalian COX11 and COX17 have been found in silico, CG9065 and CG31648, and at least one of them is expressed in S2 cells (Southon et al., 2004), there is no report addressing their role in the Cu homeostasis or in the mitochondrial function.

### Cuproenzymes and Other Cu-Related Genes

The classical enzymes described to require Cu in their activity, which have fly orthologs are: Lysyl oxidase, involved in connective tissue (Molnar et al., 2005); Peptidylglycine-α-hydroxylating monooxygenase, involved in amidation of peptides (Hwang et al., 2014); Multi-copper oxidases MCO1 and MCO3, involved in iron homeostasis (Mandilaras et al., 2013); Tyramine β-Hydroxylase, involved in neurotransmitter synthesis (Monastirioti et al., 1996); tyrosine hydroxylase, involved in synthesis of melanine and dopamine (Wright, op. 1987) and Zn/Cu superoxide dismutase (SOD1), involved in protection against oxidative stress (Phillips et al., 1989). SOD1 is by far the most studied in the fly, due to its strong relation with oxidative stress, aging and amyotrophic lateral sclerosis (ALS) (Renton et al., 2014; Casci and Pandey, 2015).

Moreover, in vivo screenings and microarray experiments have helped to identify new candidate genes involved in the regulation of Cu. Syntaxin 5 was identified in a screening of genes that modulate mortality on excess Cu (Norgate et al., 2007). It is a soluble NSF attachment protein receptor (SNARE) gene involved in intracellular vesicle trafficking with high impact on Cu uptake (Norgate et al., 2010). Other similar examples are CG14036, CG11825, CG14545 (Norgate et al., 2007). VhaPPA1-2 and bib are a couple of genes that were first discovered as required for cuticle pigmentation and lately characterized as novel mediators of Cu biology that facilitate the membrane localization of Cu transporters (Mummery-Widmer et al., 2009; Wang et al., 2014). ADP-ribosylation factor 1, Adf1, contributes to maintain the golgi structure (Southon et al., 2011). Glutamatecysteine ligase catalytic subunit gene (Gclc) is the rate-limiting enzyme in glutathione (GSH) formation. GSH is a key cellular antioxidant molecule that also plays a role in Cu regulation (Mercer et al., 2016).

## REGULATION OF Zn METABOLISM IN DROSOPHILA

Although Zn is an essential metal involved in a large panoply of key cellular processes, the in vivo physiological roles of Zn transporters are still not well characterized. In humans and in Drosophila, Zn homeostasis is mainly mediated by two large families of zinc transporters, the SLC39A or Zip (which import zinc into the cytoplasm) and the SLC30A or ZnT (which remove zinc from of the cytoplasm). Importantly, they are conserved throughout evolution from humans to flies (Lye et al., 2012). In silico and in vivo studies have identified up to 17 members of both families in the fly (Lye et al., 2013), whereas 24 proteins belong to those groups in mammals (Lichten and Cousins, 2009). In general, all Zn transporters in the fly are named by their cytological location (Yepiskoposyan et al., 2006) with the exception of catecholamines up (catsup) and fear of intimacy (foi) that retain their original names.

In the fly, most of the work to characterize the expression and function of dZip and dZnT genes has been performed in the Malpighian tubules and in the midgut because Zn mainly accumulates in the tubules and the gut is the primary site for the absorption of nutrients in Drosophila (Schofield et al., 1997). Therefore, global and systemic changes have been investigated in detail, but little is still known about local redistribution and availability of Zn within a given tissue or organ and even within the cell. Approaches based on genetic interactions have played an instrumental role in the identification of new genes involved in Zn homeostasis and in the analysis of the cellular and systemic roles of each transporter. Richard Burke's lab developed a brilliant strategy for this aim. They initially found that simultaneous overexpression of the main importer (dZip42C.1) and silencing of the main exporter (dZnT63C) triggered Zn entrapment in the cytosol, that lead to a Zn toxic phenotype (named Ztox) characterized by either retinal degeneration and loss of eye pigmentation when carried out in the developing eye (gmr-GAL4) or by scutellum loss, thorax cleft, hypopigmentation and bristles misaligned when performed in the developing thorax (pnr-GAL4) (Lye et al., 2012). Similarly, overexpression of dZnT86D or dZip71B also induce changes in the eye morphology and pigmentation (Dechen et al., 2015). In parallel, Bing Zhou's lab conducted similar studies within the gut (Qin et al., 2013; Yin et al., 2017). Importantly, all these phenotypes were modulated (rescued or worsened) by chemical or genetic manipulation of Zn levels (Lye et al., 2012, 2013; Qin et al., 2013; Dechen et al., 2015; Yin et al., 2017). The genetic interactions showed which combinations of Zn transporters modified the eye morphology, the thorax pigmentation or the response toward Zn overload/depletion. All this information along with the subcellular localization described by reporter lines has allowed dividing the Zn transporters into four subgroups.

**Table 2** and **Figure 2** recapitulate all fly genes involved in Zn transport except dZnT49B. It shows sequence similarity to the hZIP9 gene but no other information about the gene is available and it cannot be properly classified. It is important to note, that the results from the interactions between the Zn toxic genotypes and the collection of RNAi constructs provides additional evidences about the expression pattern of the Zn transporters and complements the information available in the Flyatlas (flyatlas.org). In this sense, the results already reported (Lye et al., 2012, 2013; Dechen et al., 2015) suggest that almost all transporters are expressed in the developing eye, in the thorax or in the PNS.

#### Importers of Extracellular Zn

The proteins, which have been classified as pure Zn importers into the cytosol from the extracellular matrix are dZip42C.1 (dZip1), dZip89B, dZip71B and FOI (**Figure 2**). All genes of this category are localized in the plasma membrane (van Doren, 2003; Dechen et al., 2015). dZip42C.1 has been described as the main protein responsible for dietary Zn absorption in the midgut enterocytes (Lang et al., 2012; Qin et al., 2013). In parallel to this function, dZip42C.1 also participates in Zn import into the adult nervous system (Lang et al., 2012). Unexpectedly, reduction of dZip42C.1 in the gut did not trigger any deleterious effect, even under Zn depletion conditions. This strongly suggests the presence of other dZip proteins performing Zn uptake in the gut (Qin et al., 2013). Bioinformatic analysis showed that dZip89B and dZip42C.2 (that will be introduced in the next section) were the best candidates. In agreement, dZip89B increases cytosolic Zn levels when overexpressed (Lye et al., 2013; Dechen et al., 2015). Furthermore, flies lacking dZip89B are normal, although they show reduced Zn levels in the gut along with upregulation of dZip42C.1 and dZip42C.2. In addition, dZip89B has a broader expression pattern and works as a low-affinity Zn transporter (Richards et al., 2015). All this might explain the lack of sensitivity of dZip89B mutants to Zn deprivation. dZip71B performs the

TABLE 2 | *Drosophila* proteins involved in Cu homeostasis (names according to Flybase, http://flybase.org).

*Gene symbols for human genes follow the regulation of Human Genome Organization Gene Nomenclature Committee (http://www.genenames.org). AD, Alzheimer's disease; FRDA, Friedreich's ataxia; KSD, Kidney Stones disease; PD, Parkinson's disease.*

opposite role to dZip42C.1 and its co-workers (dZip42C.2 and dZip89B). It is mainly expressed in the Malpighian tubules and imports Zn from the body for detoxification and disposal. Therefore, it is instrumental in the excretion of Zn from the organism and accordingly, silencing of dZip71B triggers Zn accumulation in the body and these flies are more sensitive to Zn overload (Yin et al., 2017). When overexpressed, dZip71B acts as a very efficient and potent Zn transporter able to create a toxic Zn accumulation on its own (Richards and Burke, 2015). Interestingly, both reports highlight cell-specific regulation of Zn metabolism.

FOI is not expressed in the gut, but is one of the cellular Zn importers in several other tissues (probably cooperating with dZip42C.1). The gene fear of intimacy (foi) showed up in genetic screens aiming to identify mutations affecting the development of fly gonads and trachea (van Doren, 2003; Mathews et al., 2005), embryonic nervous system (Pielage et al., 2004), and fly muscles (Carrasco-Rando et al., 2016). FOI is able to transport Zn in yeast and mammalian cells (Mathews et al., 2005). The Zn-related activity of foi is required for the stability of the cell-adhesion protein E-cadherin, that is essential for the proper formation of the gonads (Mathews et al., 2005, 2006). In the myoblast development, the expression of target genes of zinc-dependent transcription factors, such as Minc and Kruppel, was severely altered in foi mutants. In agreement, the phenotypes could be rescued by overexpression of other dZip genes with the same cellular localization such as dZip42C.1 and dZip71B (Carrasco-Rando et al., 2016). All these results support the role of foi as a Zn transporter and the impact of its loss-of-function in the activity of zinc-finger transcription factors.

#### Importers of Intracellular-Stored Zn

This category includes proteins that, besides having some capacity to uptake Zn from the outside of the cell, they have been shown to remove Zn also from intracellular compartments. This group encompasses the following proteins dZip42C.2 (dZip2), dZip88E, CATSUP, dZip99C, dZip48C, and dZip102B (**Figure 2**). dZip42C.2 is another Zn importer from the lumen into the enterocytes (Qin et al., 2013). Flies with reduced dZip42C.2 levels were normal but they display severe phenotypes (more than those with reduced dZip42C.1) including developmental arrest under low zinc conditions. This suggests, that dZip42C.2 might play more crucial roles than its counterpart dZip42C.1. Interestingly, expression levels of both seem to be regulated by Zn levels in the enterocytes (Qin et al., 2013). dZip88E was initially thought to be the fourth member of this group formed by dZip42C.1, dZip42C.2, and dZip89B to absorb Zn from diet (Lye et al., 2012). However, its recent characterization (Richards et al., 2017) and the results from genetic interactions (Dechen et al., 2015), suggest a completely different role. It is expressed in a small subset of cells in the gut and larval CNS displaying both, plasma membrane and intracellular localization (Dechen et al., 2015; Richards et al., 2017). Interestingly, even with this restrictive expression, mutant flies are hypersensitive toward Zn. It seems that dZip88E expression in these cells acts as a surveillance system to detect Zn toxicity and elicit a systemic response to counteract it.

Catsup is another gene that was isolated long before it was described as a dZip gene (Stathakis et al., 1999). The name of the gene comes from the high levels of catecholamines of the mutant flies due to the hyperactivity of the tyrosine hydroxylase (TH) enzyme. This result showed that Catsup is a negative regulator of dopamine synthesis. As a result, mutants display high synaptic activity and elevated mobility (Stathakis et al., 1999; Wang et al., 2011). Catsup has a very broad expression pattern (almost ubiquitous) and it is localized in the endoplasmic reticulum (ER) and Golgi apparatus (Groth et al., 2013). Accordingly, Catsup mutant flies accumulate misfolded proteins such as Notch and APPL in the Golgi apparatus and in the ER. Probably loss of catsup affects the normal processing and trafficking of proteins that, in turn, increases ER stress and caspase 3 activity (Groth et al., 2013). The genetic interactions suggest that Catsup participates in the transport of extracellular Zn and Zn efflux from Golgi apparatus (Dechen et al., 2015). Up to now, the relation of Zn with the synthesis of dopamine

and the accumulation of misfolded protein in Catsup mutants has not yet been established. dZip99C is a special case. It is classified in this group due to the results of genetic interactions (Dechen et al., 2015). However, recent research has found that it is an iron supplier and not a Zn transporter (Xiao et al., 2014). However, we cannot exclude that dZip99C is able to change Zn distribution indirectly. This would also explain the modification of Zn toxic phenotypes described in the literature (Lye et al., 2012, 2013; Dechen et al., 2015). Unfortunately, there is no functional data regarding dZip48C and dZip102B besides the information present in the FlyAtlas.

# Cellular Zinc Exporters

The proteins that act as exporters of Zn outside of the cell are dZnT63C (dZnT1) and dZnT33D (**Figure 2**). dZnT63C was first discovered in the transcriptomic analysis of the fly response to metal toxicity (Yepiskoposyan et al., 2006). It shares homology with human ZnT1 (25.6% identity and 43.1% similarity) and it is the major responsible protein for Zn import from the gut into the body (Wang et al., 2009) although it is also expressed in other tissues such as developing eye (Lye et al., 2012), testis or salivary glands (Wang et al., 2009). As expected from the proposed function, flies with reduced dZnT63C accumulated Zn in the gut and have a Zn deficiency in the rest of the fly. In agreement, these flies are also sensitive to Zn deprivation. The gut tries to compensate the loss of dZnT63C by downregulating dZip42C.1 and dZip42C.2 to reduce Zn uptake. dZnT63C is also found in the Malpighian tubules where it acts in Zn reabsorption into the body (Wang et al., 2009; Yin et al., 2017). The genetic interactions performed in the eye and in the thorax of the fly to modify Zn dyshomeostasis phenotypes (Lye et al., 2013), have classified dZnT33D as another cellular Zn exporter, although some intracellular localization seems to be present as well. However, no additional information about its cellular roles is available.

# Zinc Exporters into Cellular Compartments

This last group comprises ZnT transporters that mediate Zinc efflux from the cytoplasm into cellular compartments but that also might retain the ability of cellular export. The members of this group are dZnT35C, dZnT41F, dZnT77C, and dZnT86D (**Figure 2**). dZnT35C was first described as a gene upregulated by cadmium and Zn in flies (Yepiskoposyan et al., 2006). More importantly, it was also identified in the transcriptomic profile of dMTF-1 mutants (Yepiskoposyan et al., 2006) as one of the targets of this transcription factor. Moreover, dZnT35C is sufficient to modulate total Zn content in the fly body. Null mutations and ubiquitous downregulation of dZnT35C leads to viable individuals that are more sensitive to Zn overload (Yepiskoposyan et al., 2006; Yin et al., 2017). This phenotype is in agreement with the suggested exclusive expression of dZnT35C in the Malpighian tubules and its role in Zn detoxification and secretion from the body (Yepiskoposyan et al., 2006; Chi et al., 2015; Yin et al., 2017). Loss of dZnT35C impairs Zn detoxification and reduces, in turn, Zn tolerance. The ability of KD of dZnT35C to improve the eye degeneration induced by dZip71B (Dechen et al., 2015) also suggests neuronal expression in photoreceptor cells. Although dZnT35C has been localized in the plasma membrane, a recent and very interesting result confers dZnT35C a new location and a new role. dZnT35C is pivotal for the formation of a specialized type of lyososomal related organelles (LROs) in the Malpighian tubules that store Zn (Tejeda-Guzmán et al., 2017). These LROs might be the fly equivalent of mammalian zincosomes (Zalewski et al., 1993). The possibility to transport metals via vesicle-like structures has already been reported for mammalian ATP7A (Lutsenko et al., 2007).

dZnT86D is a Zn transporter located in the Golgi apparatus. Its overexpression is able to produce Zn toxic phenotypes in the eye without any further genetic modification (Dechen et al., 2015). Therefore, is a very interesting tool to analyse the effects provoked by Zn accumulation in the Golgi as well as changes in the intracellular Zn distribution. The ubiquitous downregulation is lethal and also produces thorax and rough eye phenotypes when performed in a tissue-specific manner (Lye et al., 2012, 2013). All this suggests that Zn plays fundamental roles in the Golgi. The last two dZnT genes, dZnT77C and dZnT41F, were classified in this category according to the results of the genetic interactions (Lye et al., 2012, 2013; Dechen et al., 2015). However, very little is known regarding their roles in the organismal and cellular Zn metabolism. On the one hand, dZnT77C is also localized in the plasma membrane of midgut cells and collaborates with dZnT63C in supplying Zn to the body from the fly intestine (Qin et al., 2013). As expected, ubiquitous silencing also enhances Zn toxicity and worsens phenotypes derived from dZnT63C KD. On the other hand, dZnT41F is also expressed in the Malpighian tubules (Chi et al., 2015; Yin et al., 2017). The similarities between silencing of dZnT41F and dZnT63C in the Malpighian tubules suggested that dZnT41F also contributes to body Zn reabsorption (Yin et al., 2017).

#### Other Genes

Additional genes that do not belong to the ZnT/Zip families have also been identified. The single Drosophila TRPM gene (dTRPM) is a curious example. Georgiev and collaborators found that dTRPM is permeable to Zn. dTRPM deficiency triggered a strong decrease in cell size that clearly resembles the effects of cellular Zn deficiency in control flies. Further experiments suggested that dTRPM regulates Zn homeostasis in a specific cellular compartment that is pivotal to growth control (Georgiev et al., 2010). Other very interesting cases are the Drosophila aquaporin homolog big brain (bib) and vhaPPA1-2, a subunit of the vacuolar-type ATPase. KD of vhaPPA1-2 or bib reduced sensitivity to high dietary zinc levels. Their study showed loss of these two genes altered the correct subcellular localization of zinc transporters. The defect in this new level of control of Zn homeostasis was probably due to a failure in the endosomal recycling of proteins back to the membrane that hence reduced the uptake of Zn (Wang et al., 2014). Very recently, kuzbanian (kuz) a gene that was already described as a target of dMTF-1 (Yepiskoposyan et al., 2006) has been found to confer tolerance to Zn when overexpressed (Le Manh et al., 2017).

The unexpected finding of abnormal Zn levels in a heterozygous fumble mutant (Gutiérrez et al., 2010) lead to

the identification of an unknown recessive factor on the X chromosome displaying a strong influence on the organismal Zn content (Afshar et al., 2013). This mutation has been named pocozinc in the unpublished observations of Fanis Missirlis'group (Tejeda-Guzmán et al., 2017). Although no definitive evidence was presented, all results point toward the white gene as the responsible for the poco-zinc phenotype. Following this initial discovery, authors realized that other mutants of genes involved in the transport of eye pigments, such as scarlet (st) or carmine (cm), also display reduced Zn levels. It seems that the collective action of most of these proteins is required for the biosynthesis of Zn storage granules in the Malpighian tubules of Drosophila (Tejeda-Guzmán et al., 2017) and thus to maintain systemic Zn homeostasis. Remarkably, the process of characterization of the poco-zinc locus has also highlighted the crucial importance of controlling the genetic background of flies in order to avoid misinterpretation of results when analyzing Zn metabolism and comparing different genotypes. Finally, in a recent publication from Norbert Perrimon's lab, a Drosophila cell-based RNAiscreen has allowed the identification of novel genes conferring sensitivity or protection toward Zn such as CG11897 (red dog mine, rdog) and some disease-related genes such as IA-2 protein tyrosine phosphatase, an ortholog of human PTPRN or CG32000, a putative ortholog of human ATP13A2 (Mohr et al., 2017).

## THE ROLE OF Cu AND Zn IN DROSOPHILA MODELS OF HUMAN NEURODEGENERATIVE DISEASES

Cu also exerts some specific roles in the nervous system. Cu is necessary in flies and mammals for the biosynthesis of neurotransmitters (Sellami et al., 2012; Opazo et al., 2014) and thus, for brain activity. In addition, Cu modulates and even blocks the activity of receptors involved in neurotransmission such as NMDA, AMPA or GABA. It has been suggested that these synaptic roles are mediated by interplays between Cu and proteins like the amyloid precursor protein (APP), the prion protein (PrP), α-synuclein (α-syn) or neurotrophic factors. Interestingly, PrP proteins seem to be target genes of MTF-1 (Grzywacz et al., 2015). Moreover, Cu levels are altered in brains of patients suffering from AD, Huntington's Disease (HD) and PD. In AD and PD, Cu levels are overall reduced but enriched in the amyloid plaques, whereas Cu accumulates in HD brains (Fox et al., 2007; Davies et al., 2016; McAllum and Finkelstein, 2016). Cu binds directly bind to α-syn and promotes the formation of toxic oligomers (Davies et al., 2016). Similarly, Cu has also been found to facilitate amyloid beta (Aβ) deposition, formation of tau fibrills (McAllum and Finkelstein, 2016) as well as aggregation of Huntingtin (Htt) containing aberrant polyQ expansions (Fox et al., 2007). All these evidences and many more (Opazo et al., 2014; D'Ambrosi and Rossi, 2015) reveal new connections between Cu and neurodegenerative diseases. The critical role of Cu in several neurodevelopmental, neuromuscular and neurodegenerative disorders has been recently corroborated with the characterization of the cellular interactome of human ATP7A (Comstra et al., 2017). Similarly, Zn is also concentrated in glutamatergic synaptic terminals where it interacts with neurotransmitter receptors to modulate synaptic activity (Sensi et al., 2009, 2011). Although evidences about Zn levels in AD brains are contradictory, Zn metabolism is clearly disturbed in AD and several studies have demonstrated that Zn is crucial in the olimerization of Aβ. Furthermore, it has been suggested that combination of Cu and Zn dyshomeostasis increased toxicity of Aβ (Sensi et al., 2011; McAllum and Finkelstein, 2016). Mutations in ATP13A2 (PARK9) have been associated to PD. ATP13A2 encodes a P5B-type ATPase and although its specific substrate is still not clear, some evidences point toward Zn as a very good candidate (Tsunemi and Krainc, 2014).

#### Cu-Related Diseases

Menkes disease (MD) is the most important and best characterized neurological disorder related to Cu homeostasis. MD is a pediatric fatal metabolic syndrome accompanied with neurodevelopmental and neurodegenerative defects with a prevalence of 1:50,000 individuals (de Bie et al., 2007). MD is a recessive disorder produced by a loss of function of ATP7A. In the last 50 years, more than 370 mutations affecting ATP7A have been described. Depending on the severity of the symptoms, they have been ascribed, to different types of Xlinked disorders named classical MD, occipital horn syndrome and spinal muscular atrophy (Zlatic et al., 2015). However, the mechanism of neurodegeneration is still poorly understood. Impairment of ATP7A functions induces accumulation of Cu into intestinal enterocytes but a deficiency of Cu in plasma, kidney, neurons and astrocytes due to the lack of transport of Cu into these tissues (Kaler, 2011). Mutations in ATP7A triggers several neurological phenotypes such as abnormal neuroblast migration, atrophy of gray and white matter as well as loss of neurons in the brain cortex and aberrant dendritic arborizations (Zlatic et al., 2015). The so called "oligoenzymatic hypothesis" (lack of Cu in cuproenzymes) does not explain the constellation of neurological manifestations and therefore, other proteins must be involved, too. In this sense, the existing mouse models very nicely recapitulate the human physiological and cellular defects (Lenartowicz et al., 2015) but they have failed to unveil new elements of the pathogenic mechanism. Moreover, it is still a matter of debate whether neurodegeneration in Menkes is a consequence of loss of ATP7A in neurons or of nutritional Cu. The potential of the fly will be very helpful to address particular roles of Cu in neurological disorders as well as to unveil novel neuronal specific proteins involved in Cu homeostasis and in the pathogenesis of MD.

As stated above, DmATP7 is the fly ortholog of ATP7A (Southon et al., 2004). Remarkably, null alleles of DmATP7 resemble human MD phenotypes (Norgate et al., 2006) and DmATP7 is able to restore normal copper levels in MD patient's fibroblasts (Southon et al., 2010). All this shows a clear functional homology between human and fly proteins. Analysis of mutants shows that DmATP7 is essential in early development as mutants never reach third instar larvae. Mutant larvae also display hypopigmentation (due to lack of Tyr activity) and lethargic behavior (because of compromised synthesis of neurotransmitters). Therefore, DmATP7 plays a dual role

exporting Cu from the cells and delivering it to cuproenzymes (Norgate et al., 2006). Silencing of DmATP7 in the fly digestive tract is sufficient to induce Cu retention in the gut (Binks et al., 2010) and to reduce Cu content in the nervous system. This systemic effect triggers, in turn, neurodevelopmental Menkeslike phenotypes such as enhanced pupal lethality and smaller brain size (Bahadorani et al., 2010a). However, the small proportion of adults that successfully completed development show normal lifespan, but with hypersensitivity to oxidative insult (Bahadorani et al., 2010a) likely because lack of a functional antioxidant enzyme Cu/ZnSOD. Reduction of DmATP7 in different neuropeptidergic neurons had a strong impact on amidation of several neuropeptides but effects on fly behavior were more limited than anticipated (Sellami et al., 2012). DmATP7 has recently been shown to interact with the oligomeric Golgi complex (COG) to control the development of synapsis via a novel mechanism (Comstra et al., 2017). All these results indicate that DmATP7 plays cell and non-autonomous roles in Cu homeostasis. Moreover, survival of flies lacking DmATP7 is improved by increasing Cu content in the food and by upregulating dMTF-1. This will consequently promote expression of Ctr1B to potentiate supply of Cu and of Mtns to reduce toxic effects of Cu accumulation in the gut (Bahadorani et al., 2010a). It is important to highlight that Drosophila only contains one ATP7 ortholog and therefore the fruit fly might also be an interesting organism to model Wilson's disease (WD), a human disorder caused by mutations in the ATP7B gene which produces increased intracellular Cu levels, accumulation of ROS, mitochondrial dysfunction and cell death (Bandmann et al., 2015). The fly models of MD and WD have gone one step beyond since they are being used as diagnostic tools to establish the pathogenicity of variants found in patients. The ability of given variants to improve the larval lethality of the fly null mutant helps to discriminate between pathological and control-like variants. In addition, subcellular localization of mutant forms have provided insight into the mechanism of copper dyshomeostasis in MD and WD patients (Mercer et al., 2017).

Several other neurodegenerative disorders have been shown to have an intimate relation with Cu (Ayton et al., 2013; McAllum and Finkelstein, 2016), although it is not clear if Cu is a friend or a foe. Usually WD patients manifest Parkinson-like symptoms (Bandmann et al., 2015). Therefore, it would be interesting to test the influence of Cu in PD models and whether manipulation of cellular Cu content might have a beneficial effect (**Table 1**). From the whole plethora of fly models (Botella et al., 2009), this has only been addressed in flies deficient for the protein Parkin (Saini et al., 2010). In this work, Cu-specific chelation by means of BCS was sufficient to increase longevity of parkin null mutants. Additional evidences suggested that reduction of ROS was mediating the neuroprotective effect of BCS in parkin deficient flies (Saini et al., 2010). It is likely that the combination of redox-activity of free Cu and the mitochondrial dysfunction in parkin mutant flies boosted the generation of ROS in these flies. The Cu-parkin relation was further corroborated by genetic interactions. Indeed, loss of parkin exacerbated the retinal degeneration triggered by overexpression of the Cu importer Ctr1B (Hua et al., 2010). Cu levels have been demonstrated to be lowerin PD patients (Davies et al., 2016) although the rescue with BCS from Saini and collaborators goes in an opposite direction. Therefore, it would be of high relevance for the scientific community to study the impact of Cu biology in other PD fly models.

Modulation of Cu metabolism has also successfully improved the deleterious defects of toxic Aβ42 peptides in Drosophila models of AD (Lang et al., 2013; Singh et al., 2013; **Table 1**). Aβ proteins are the major component of plaques in AD patients. The Aβ42 isoform is more prone to aggregation due to the 2 extra aminoacids at the C-terminus (White et al., 1999). Reduction of cellular Cu content by silencing the expression of Ctr1B and Ctr1C (Lang et al., 2013), by overexpressing DmATP7 (Lang et al., 2013) and dMTF-1 or MtnA (Hua et al., 2011b) improved neurodegeneration, locomotion, longevity and oxidative stress markers. Similar effects were obtained by chemical chelation of Cu (Hua et al., 2011b; Singh et al., 2013). On the contrary, increasing cellular Cu or impairing intracellular Cu delivery worsened the phenotypes (Sanokawa-Akakura et al., 2010; Hua et al., 2011b). The work from Lang and collaborators raised a couple of interesting and paradoxical questions. First, AD phenotypes are rescued by panneuronal silencing of Ctr1B and C, genes supposed to be only expressed in fly gut and male gonads (Zhou et al., 2003; Hua et al., 2010). This indicates that better reporters to study expression patterns of these genes are needed. Second, the improvements were accompanied by increased amounts of higher-molecular weight forms of Aβ42. This result clearly questions the toxicity of the aggregates. However, similar genetic interventions in a fly model of HD expressing the mutant form of Htt (expanded polyQ tract) successfully reduced the levels of PolyQ aggregation by decreasing Cu content in flies (Xiao et al., 2013). Therefore, Cu metabolism might exert different and even opposite roles in distinct disease models. As a consequence, locomotion, survival and brain degeneration were also improved in this HD fly model (Xiao et al., 2013). These experiments revealed that Cu facilitates protein aggregation in HD. In agreement, a mutant form of Htt unable to bind Cu displayed a reduced toxicity (Xiao et al., 2013).

Other reports in the fly have identified genes with influence on neurodegenerative or neuropsychiatric diseases that resulted in new actors of Cu metabolism. Presenilins participate in the processing of Aβ proteins as they are the catalytic unit of the γ-secretase complex and their mutations underlie the majority of early-onset AD cases. Interestingly, secretase-independent roles have been reported for presenilins (Stiller et al., 2014). Silencing of PSN, the presenilin ortholog present in flies, in the fly gut reduced copper levels in flies and increased tolerance to excess dietary copper but also susceptibility to oxidative insult. PSN is suggested to contribute to the localization of Cu importers Ctr1A and Ctr1B in the plasma membrane (Southon et al., 2013b) but the exact action mechanism still needs to be completely elucidated. Misexpression of high molecular weight immunophilin FKBP52, which is a known interactor of the Cu chaperone Atox1, also modulates toxicity of Aβ42 (Sanokawa-Akakura et al., 2010). Finally, disruption of the dysbindin/BLOC-1 complex in the fly by means of Drosophila BLOC-1 mutants is sufficient to alter Cu metabolism suggesting that external factors might modulate schizophrenia via mechanisms that are conserved from humans to flies (Gokhale et al., 2015).

#### Zn-Related Diseases

Zn also needs to be tightly regulated because excess or deprivation of Zn has severe health effects. Many reviews summarize the toxic consequences of Zn dyshomeostasis in humans and murine models (Fukada and Kambe, 2011; Hagmeyer et al., 2014; Kambe et al., 2014, 2015; Terrin et al., 2015). For example, Zn deficiency in the fetus affects organ growth that can result into preterm birth or even early death (Terrin et al., 2015). In mouse models, several behavioral tests have linked Zn deficiency with deficits in learning and memory or increased anxiety or depression-like disorders (Hagmeyer et al., 2014). Zn homeostasis is a pretty new field in the fly and although several new tools are being continuously implemented to further characterize the Drosophila Zn transporters, there are not many fly models of Zn-dependent diseases. In humans, mutations or even Single Nucleotide Polymorphisms (SNPs) in ZnT or Zip genes have been already associated to particular diseases (Fukada and Kambe, 2011; Kambe et al., 2015). For example, mutations in ZnT10 are responsible for hepatic cirrhosis that is accompanied with Parkinsonism or mutations in Zip4 triggers Acrodermatitis enteropathica. On the other hand, SNPs in ZnT8 are linked with increased susceptibility toward type I and II diabetes mellitus. From all human disorders directly related to Zn homeostasis, the Spondylocheirodysplasia-Ehlers-Danlos Syndrome-Like is the only one modeled in Drosophila (Xiao et al., 2014). The Ehlers-Danlos Syndrome is produced by loss of function of the human Zip13 transporter. However, the Drosophila ortholog dZip99C (CG7816) is involved in iron efflux and not in Zn transport. Loss of dZip99C affects iron storage by ferritin, increases cytosolic iron and, in agreement with the human counterpart, reduces collagen production. Interestingly, the fact that hZip13 but not hZip7 (a closely related gene that has been shown to import Zn) can complement dZip99C (Xiao et al., 2014), might suggest that the human disorder is also triggered by iron deregulation and thus, Zn defects are a secondary event.

Manipulation of Zn regulatory network has been a very successful approach to ameliorate neuronal dysfunction in Drosophila models of AD as well as to decipher the functional roles of Zn in this disease (**Table 2**). In an AD model based on overexpression of a mutant version of human Tau, results from a genetic screening showed that increasing or reducing the expression of a dZip42C.1 and dZnT63C successfully modified Tau-related phenotypes in the fly (Huang et al., 2014). Reduction of Zn content by dZnT63C overexpression, silencing of dZip42C.1 or chemical chelation improved longevity, brain vacuolization and retinal degeneration. Remarkably, authors proved in the fly that Tau phosphorylation was mediated by Zn and that hyperphosphorylation was a cardinal mechanism underlying toxicity. Furthermore, as Zn can directly bind Tau protein, authors mutated specific residues to suppress this interaction. This was sufficient to reduce Tau toxicity by suppressing Tau aggregation but without changing Tau phosphorylation. Thus, this work showed in vivo two Zn-dependent mechanisms underpinning Tau toxicity (Huang et al., 2014). In a second AD model, Zn chelation was sufficient to improve longevity, locomotion and photoreceptor degeneration in flies overexpressing Aβ42 protein. As expected, Zn supplementation exacerbated the phenotypes. This enhancement was suppressed in a special variant of Aβ42 in which Zn binding was abolished (Hua et al., 2011b). In agreement with the critical role of Zn metabolism in this model, other authors found that dZip42C.1 level increases with age in the brain of Aβ42-expressing flies, whereas it goes down in controls. Such levels explain the accumulation of Zn in the brain of Aβ42 (Lang et al., 2012) and the toxic effects of Zn supplementation (Hua et al., 2011b). In line with these evidences, KD of dZip42C.1 was sufficient to reduce neurodegeneration, prolong lifespan and improve memory loss as well as Aβ42 deposits in transgenic flies (Lang et al., 2012). All these results suggest that dZip42C.1 is critically involved in the pathology of Aβ42. Paradoxically, overexpression of dZip42C.1 also resulted in obvious memory recovery but the mechanism was not described in the manuscript.

Similar contradictory findings have been recently described in a Drosophila model of Friedreich's ataxia (FRDA). FRDA is the most important recessive ataxia in the Caucasian population. Impairment of transcription of the gene frataxin is the molecular cause underlying the disease (Campuzano et al., 1996). Most of the current evidences support a strong relation between frataxin and iron in several models (Kakhlon et al., 2008; Huang et al., 2009; Schmucker et al., 2011), including the fly (Soriano et al., 2013; Navarro et al., 2015; Chen et al., 2016). Moreover, genetic and chemical manipulation of iron biology was found to have a positive impact in FRDA phenotypes (Kakhlon et al., 2008; Whitnall et al., 2008; Navarro et al., 2015). Remarkably, flies displaying reduced levels of frataxin also seemed to have altered levels of other metals such as Zn and Cu. Although the contribution of other metals was already suggested by studies in human samples (Koeppen et al., 2012, 2013), this fly work was the first one showing that therapies based on such metals might be beneficial (Soriano et al., 2016). Indeed, Zn and Cu chelators improved frataxin deficiency without altering iron content. In this line, silencing either dZip42C.1, dZip42C.2 and dZip88E or dZnT35C, dZnT41F and dZnT63C also improved FRDA conditions via reduction of the iron content (**Table 2**). All these results raise several interesting questions: Why are Cu and Zn accumulating in FRDA flies? Why does KD of genes with opposite function or acting in different cellular compartments trigger the same effect? Did they also modify the accumulation of Zn in FRDA flies? Is it possible that any of these transporters has also a mitochondrial function?

Studies of a Drosophila PD model took advantage of the antioxidant properties of Zn and explored the possibility to counteract the increased oxidative stress in the fly parkin mutant (Greene et al., 2005) by Zn supplementation. Interestingly, parkin mutant flies displayed altered levels of several Zn transporters such as dZnT35C, dZnT63C, or foi and had reduced Zn content in the head. Importantly, dietary Zn successfully increased eclosion frequency, survival rate and head zinc content of parkin mutants (Saini and Schaffner, 2010). Results showed that Zn boosted expression of Mtns and that the ROS scavenging properties of Mtns mediated the rescue mechanism. In relation to PD, Catsup mutants, surprisingly, have increased tolerance against paraquat induced neurotoxicity (Chaudhuri et al., 2007). This result is interesting since Catsup mutants display high tyrosine hydroxylase (TH) activity and therefore increased dopamine pools (Stathakis et al., 1999), whereas reduction of TH expression and dopamine levels rescued PD phenotypes in two other fly models of the disease (Bayersdorfer et al., 2010). Although the mechanism driven by loss of Catsup was not addressed in the manuscript, we can speculate that the activation of stress pathways in the mutants (Groth et al., 2013) might contribute to counteract the paraquat effect.

In the literature, the relation between Zn and fly models of human diseases is mostly based on studies about neurodegenerative disorders (**Table 2**). Importantly, the fly has also been useful to mimic other human diseases and expand their knowledge beyond the state of the art. A recent report showed that Zn is also a crucial mediator in the formation of urinary stones. In human, deficiency of xanthine dehydrogenase is responsible of such kidney stones (Arikyants et al., 2007). In the fly, silencing of the ortholog gene (Xdh also known as ry, CG7642) also induces the presence of small stones in the intraluminal content of Malpighian tubules. Spectrometric analysis of such stones revealed high similarity to the human ones including the presence of abundant Zn. In line with this novel perspective, reduction of the expression of three ZnT transporters (dZnT35C, dZnT41F, dZnT63C) and of dZip71B that are highly expressed in the Malpighian tubules mitigated the formation of stones as well as other phenotypes associated to loss of Xdh (Chi et al., 2015; Yin et al., 2017). Evidences suggest that downregulation of these genes avoid the accumulation of Zn in the lumen of the tubules. In agreement, Zn chelation by N,N,N'N'-tetrakis- (2-pyridylmethyl)ethylenediamine (TPEN) also lead to similar results (Chi et al., 2015). A link between cancer and Zn has been established throughout the processing of the morphogen hedgehog (hh). Zn normally inhibits fly hh autoprocessing and thus upon Zn deficiency there is an overactivation of the pathway that might contribute to pathogenesis of several cancer types (Xie et al., 2015).

#### THE POTENTIAL OF DROSOPHILA FOR FUTURE STUDIES

However, there are some aspects of Cu and Zn biology that still remain obscure. For example, it is of paramount importance to understand the need of so many ZnTs and Zips to regulate Zn metabolism. The number of genes performing similar functions confers an unexpected level of complexity. Furthermore, establishing the precise spatial and temporal expression pattern

#### REFERENCES

of each gene regulating Cu and Zn homeostasis will facilitate the interpretation of many aspects of the metal biology. Frank Schnorrer's and Hugo Bellen's labs have created two large-scale transgenic collections that allow protein visualization. The first one is based on a fosmid library of clones in which target genes are C-terminally tagged within their genomic context (Sarov et al., 2016). The second one has modified the Minos mediated integration cassette (MiMIC) to introduce a dominant marker and a gene-trap cassette flanked by two inverted 8C31 attP sites (Venken et al., 2011). The MiMIC collection also allows the replacement of the gene-trap cassette via recombinase mediated cassette exchange (RMCE) even without the need of microinjection (Nagarkar-Jaiswal et al., 2015a,b). A similar approach combining CRISPR and RMCE has been reported (Zhang et al., 2014). The labs of Christopher Potter, Matthias Landgraf and Benjamin White have very nicely mixed both, MiMIC, and CRISPR, strategies and have generated another collection of so called Trojan-GAL4 lines (Diao et al., 2015). This strategy takes advantage of MiMIC transposons located in introns between coding exons and exchanges their content with a T2A-GAL4 cassette. Because of it, the native gene product will be cleaved and translation of GAL4 will occur as an independent protein. A perfect complement to these reporters in the possibility to perform cell-type-specific transcriptional profiling without cell isolation (Southall et al., 2013; Marshall et al., 2016). This extremely powerful tool developed in Andrea Brand's lab might help to clarify the cells/tissues expressing the different Zn and Cu transporters.

Up to now, tissue-specific analysis of Cu and Zn relies on RNAi lines. However, they are not completely effective and the residual gene activity might be sufficient to hide some important aspects of gene function. To bypass this, Simon Bullock's lab has developed a fast approach to generate CRISPR mutants by means of the UAS/GAL4 system (Port and Bullock, 2016). Finally, the possibility to perform unbiased genetic screens, which confers Drosophila an important advantage over other higher eukaryotic model systems, will allow to identify new genetic factors involved in the regulatory networks of Cu and Zn and modifiers of phenotypes in the fly models of human diseases.

#### AUTHOR CONTRIBUTIONS

JAN and SS designed, wrote and approved the final manuscript.

#### ACKNOWLEDGMENTS

JAN and SS thank Olga Rivero for critically reading the manuscript and Maribel Calabuig and Ursula Roth for helping with the Art Work and figure preparation.

Afshar, N., Argunhan, B., Bettedi, L., Szular, J., and Missirlis, F. (2013). A recessive X-linked mutation causes a threefold reduction of total body zinc accumulation in Drosophila melanogaster laboratory strains. FEBS Open Bio. 3, 302–304. doi: 10.1016/j.fob.2013.07.003

Al-Momani, F. A., and Massadeh, A. M. (2005). Effect of different heavy-metal concentrations on Drosophila melanogaster larval growth and development. Biol. Trace Elem. Res. 108, 271–277. doi: 10.1385/BTER:108:1-3:271

Arikyants, N., Sarkissian, A., Hesse, A., Eggermann, T., Leumann, E., and Steinmann, B. (2007). Xanthinuria type I: a rare cause of urolithiasis. Pediatr. Nephrol. 22, 310–314. doi: 10.1007/s00467-006-0267-3

suppress FRDA phenotypes in a Drosophila model of the disease. PLoS ONE 11:e0159209. doi: 10.1371/journal.pone.0159209

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Navarro and Schneuwly. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

#### Zvonimir Marelja<sup>1</sup> , Silke Leimkühler <sup>2</sup> and Fanis Missirlis <sup>3</sup> \*

1 Imagine Institute, Université Paris Descartes—Sorbonne Paris Cité, Paris, France, <sup>2</sup> Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany, <sup>3</sup> Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México, Mexico

#### Edited by:

David W. Killilea, Children's Hospital Oakland Research Institute, United States

#### Reviewed by:

Aram Megighian, Università Degli Studi di Padova, Italy María Dolores Moltó, Universitat de València, Spain

> \*Correspondence: Fanis Missirlis<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 25 October 2017 Accepted: 16 January 2018 Published: 14 February 2018

#### Citation:

Marelja Z, Leimkühler S and Missirlis F (2018) Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism. Front. Physiol. 9:50. doi: 10.3389/fphys.2018.00050 Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.

Keywords: aldehyde oxidase, DNA polymerase, electron transport chain, ecdysone, iron regulatory protein, quiescent mitochondria, magnetoreceptor, mitoflashes

#### INTRODUCTION

In the first known biochemical reactions on earth, molybdenum and iron-sulfur (Fe-S) clusters enabled electron transfers turning inorganic molecules into hydrogenated carbon molecules (Mortenson, 1964; Eck and Dayhoff, 1966; Hall et al., 1971; Ochiai, 1978; Wächtershäuser, 1988; Russell and Martin, 2004; Zhang and Gladyshev, 2008; Nitschke and Russell, 2009; Schoepp-Cothenet et al., 2012; Stüeken et al., 2015). Similar biochemistry remains active in living organisms carried out by a variety of metallo-enzymes. In this hypothesis and theory article, we present examples of Fe-S and molybdenum cofactor (Moco) enzymes from the dipteran fly Drosophila melanogaster, a genetically amenable and thoroughly characterized experimental model system (Bellen et al., 2010; St Johnston, 2013; Mohr et al., 2014). By looking at their multiple physiological functions, we propose that Fe-S enzymes are central in the development, life cycle transitions and aging of flies. Given the conservation of these phenomena in the evolution of the animal kingdom, we anticipate that many of our descriptions will be transferable to other organisms.

In the first part of the article, we present how Fe-S clusters are formed, a process that has been studied extensively by biochemists in prokaryotes (Roche et al., 2013; Blanc et al., 2015), yeast (Martinez-Pastor et al., 2017), plants (Balk and Schaedler, 2014), and humans (Paul and Lill, 2015; Rouault and Maio, 2017), but also by the biomedical community intent to find a therapy for patients with Friedreich's ataxia, caused by reduced expression of the frataxin (FXN) gene (Campuzano et al., 1996). Other Fe-S proteins are also implicated in human disease (Rouault, 2012; Beilschmidt and Puccio, 2014; Isaya, 2014). In D. melanogaster, the pioneering work of Maria Moltó and coworkers has almost exclusively focused on the Drosophila frataxin homolog describing what goes wrong when Fe-S biosynthesis is disrupted in flies (reviewed in Mandilaras et al., 2013; Tang and Zhou, 2013b; Zhu et al., 2014; Calap-Quintana et al., 2017). Furthermore, we describe the biosynthesis of Moco (Rajagopalan, 1997; Mendel and Leimkühler, 2015; Leimkühler, 2017), whose basic structure has two sulfur atoms of the tricyclic pyranopterin molecule molybdopterin (MPT) coordinating the Mo atom (Rajagopalan et al., 1982). Work on Moco enzymes in Drosophila started in the fifties and Victoria Finnerty studied the Moco biosynthetic pathway during the last quarter of the twentieth century (Kamdar et al., 1997). Her research program used the molybdoenzyme xanthine dehydrogenase (Xdh) encoded by the rosy gene, whose activity is required for the formation of eye pigments for reasons that are still not fully resolved (Phillips and Forrest, 1980; Wiley and Forrest, 1981; Ferre et al., 1986; Keith et al., 1987; Hilliker et al., 1992), and therefore mutants affecting Moco biosynthesis have evident eye color phenotypes (Kamdar et al., 1997). We complement this review with new discoveries in the role of proteins involved in Fe-S cluster and Moco biosynthesis by showing the original data, which are not published elsewhere (Marelja, 2013), and with a new hypothesis to explain previous observations that dietary copper decreases iron storage in Drosophila (Poulson and Bowen, 1952; Bettedi et al., 2011).

In the second part of the article, we review studies of the Drosophila molybdoenzymes Xdh, aldehyde oxidase (Aox), sulfite oxidase (Suox). Special attention is paid to the problem of how Xdh, a cytosolic enzyme that requires two Fe-S clusters, Moco and flavin for activity (Hughes et al., 1992; Doyle et al., 1996), finds its way into pigment granules of the eye, the only enzyme with such cofactors known to reside in the endomembrane system (Reaume et al., 1989, 1991). Our hypothesis is that exosomes are involved in the process. The fly literature on mitochondrial Fe-S enzymes required for respiration and the biosynthesis of ecdysone, heme, and lipoate is summarized. The role of cytosolic and nuclear Fe-S enzymes in DNA replication, transcription and translation is also reviewed, followed by a brief discussion of the regulation of the Fe-S cluster of iron regulatory protein-1A (IRP-1A) and cellular iron sensing in Drosophila. We then move to the question of how mitochondria sense iron, where we present a new hypothesis suggesting that the mitochondrial superoxide dismutase (Sod2) is a possible mitochondrial iron sensor. Our model of mitochondrial iron sensing also explains the previously observed superoxide bursts in mitochondria (Wang et al., 2008) and the connection between mitochondrial Fe-S cluster biosynthesis and lipogenesis (Tong and Rouault, 2007). Last, we revisit the question of whether Fe-S and/or Moco enzymes are involved in the circadian clock (Mandilaras and Missirlis, 2012).

In the third part of the article, we communicate how profoundly cell physiology depends on Fe-S enzymes. We review the shifts in cell metabolism from glycolysis to aerobic respiration during development (Tennessen et al., 2011, 2014) and during stem cell differentiation (Homem et al., 2014; Sieber et al., 2016), emphasize the requirement of Fe-S clusters for growth through the larval stage and into metamorphosis (Anderson et al., 2005; Uhrigshardt et al., 2013; Llorens et al., 2015; Palandri et al., 2015) and the decline of mitochondrial respiration during aging (Vann and Webster, 1977; Yan et al., 1997; Ferguson et al., 2005). We then discuss the interesting finding that in the female germ line, stem cell differentiation requires the mitochondrial ATP synthase, but not the respiratory chain enzymes (Teixeira et al., 2015). We critically evaluate the possibility that the presence of quiescent mitochondria in the female germ line may serve as a protective hereditary mechanism against the accumulation of mutations in their genome (Allen, 1996). Finally, we describe specific functions of Fe-S or Moco enzymes in the major organs of the fly, concluding that they are a biochemically active component of the complex organization that characterizes living animals.

## Fe-S CLUSTER AND MOCO BIOSYNTHESIS

Fe-S cluster assembly initiates in the mitochondrial matrix. We summarize the different steps of the assembly process (**Figure 1**); for detailed reviews of the pathway the reader is referred elsewhere (Roche et al., 2013; Balk and Schaedler, 2014; Blanc et al., 2015; Paul and Lill, 2015; Martinez-Pastor et al., 2017; Rouault and Maio, 2017). The mitoferrin transporter ensures mitochondrial iron uptake, whereas the L-cysteine desulfurase Nfs1 provides the inorganic sulfide as persulfide. Electrons are required for the cleavage of the persulfide group and assembly of the cluster, which are supplied from ferredoxins Fdx1 and Fdx2. The first assembly protein is IscU, which can accommodate [2Fe-2S] or [4Fe-4S] clusters. Fe-S clusters are transferred from IscU to other scaffold proteins like IscA1, IscA2, Iba57, Nfu, BolA1, or BolA2 that show specificity of cluster delivery to the target enzymes. For Fe-S cluster transfer between the assembly proteins, or between assembly proteins and target enzymes, specialized

FIGURE 1 | Subcellular compartmentalization of the Fe-S cluster and Moco assembly processes. Iron and sulfur from cysteine are assembled on IscU in a process that requires electrons provided by ferredoxins (Fdx1, Fdx2, which are themselves Fe-S proteins). Chaperones and Grx5 are required for transfer of the Fe-S cluster to further scaffold proteins. The Drosophila homologs were identified by Blast searches, proposed names for the gene products (in white boxes) were based on the corresponding nomenclature in humans, current Flybase annotations are shown in blue font. Our own results (Marelja, 2013) confirm that the Drosophila genes CG12264 and CG3717 are the homologs of the human genes NFS1 and ISD11, respectively. Multiple Fe-S enzymes are present in mitochondria, cytosol, and nucleus, but so far none have been reported in the secretory pathway. All known components of the CIA pathway are conserved in Drosophila, but the first steps of CIA remain to be elucidated. Moco biosynthesis initiates in the mitochondria with the Fe-S protein Mocs1 and continues in the cytosol with Mocs2, Mocs3, and Cinnamon producing Moco used in Suox. Maroon-like sulfurates Moco for Xdh and Aox. Mitochondrial and cytosolic forms of the cysteine desulfurase Nfs1 (yellow box) provide sulfide to Fe-S cluster and Moco biosynthesis, respectively.

chaperones (Hsc20 and its cognate partner) and glutaredoxin-5 (Grx5) are required. A cytosolic Fe-S cluster assembly (CIA) pathway has been described, however it is unclear how the first clusters are formed on the CIA complex. The cytosolic electron donors cytokine-induced apoptosis inhibitor-1 (CIAPIN1) and glutaredoxins Grx3 and Grx4 have been identified (**Figure 1**). Only three genes involved in Fe-S biosynthesis have been individually studied in flies: (i) Drosophila frataxin whose exact biochemical function is still unclear (Cañizares et al., 2000; Anderson et al., 2005, 2008; Llorens et al., 2007; Kondapalli et al., 2008; Runko et al., 2008; Navarro et al., 2010, 2011, 2015; Shidara and Hollenbeck, 2010; Soriano et al., 2013, 2016; Tricoire et al., 2014; Calap-Quintana et al., 2015; Palandri et al., 2015; Chen et al., 2016b; Edenharter et al., 2017), (ii) IscU that encodes a protein assembly platform for Fe-S cluster biosynthesis (Dzul et al., 2017), and iii) Hsc20 encoding one of two chaperones that mobilize the Fe-S cluster from IscU to downstream Fe-S proteins in the mitochondria (Uhrigshardt et al., 2013).

#### The Drosophila Nfs1/Isd11/IscU/Frataxin Complex

Two Drosophila Fe-S cluster assembly proteins have been purified to date, frataxin (Kondapalli et al., 2008) and IscU (Dzul et al., 2017). We characterized the core mitochondrial protein complex involved in Fe-S cluster assembly in flies (Marelja, 2013). Based on previous work with the human L-cysteine desulfurase NFS1 (Marelja et al., 2008, 2013), the Drosophila Nfs1 homolog (CG12264) was purified. The human and Drosophila enzymes share an amino acid sequence identity of 78% when the mitochondrial targeting sequences are removed from the calculation. All amino acids implicated in enzymatic function of the human protein are conserved in Drosophila Nfs1 (**Figure 2**).

The first 53 amino acids of the full length Drosophila Nfs1 were removed from an Escherichia coli-driven protein expression construct (for detailed Materials and Methods see Marelja, 2013).  The respective Nfs111–53/Isd11 and Nfs111–53/IscU complexes were affinity purified and recovered in a stable, soluble form. Higher molecular weight complexes were obtained when all three Drosophila proteins were co-expressed and also when human FXN (a gift from Kuanyu Li; Xia et al., 2012; Friemel et al., 2017) was added to the Nfs111–53/Isd11/IscU complex (**Figure 3A**). Our own efforts to purify Drosophila frataxin as in Kondapalli et al. (2008) were unsuccessful (Marelja, 2013).

The characteristic yellow color observed for other L-cysteine desulfurases containing pyridoxal 5′ -phosphate (PLP) as a prosthetic group was confirmed in absorption spectra of Drosophila Nfs1 at 420 nm (**Figure 3B**, solid black lines on both panels). Moreover, addition of the enzyme's substrate L-cysteine induced a decrease of absorbance at 420 nm and an increase of absorbance at 320 nm (**Figure 3B**, solid and dashed red lines), as reported for Azotobacter vineladii L-cysteine desulfurase NifS, showing that the α-amino group of cysteine binds to the PLP at the enzyme's active site (Zheng et al., 1993). For Nfs111–53/Isd11, L-cysteine binding reached equilibrium at 1 min (**Figure 3B**, top panel). In contrast, binding of L-cysteine to the Nfs111–53/Isd11/IscU complex was slower (**Figure 3B**, bottom panel), suggesting that the presence of IscU reduced substrate accessibility to Nfs1 in vitro. The Drosophila Nfs111–53/Isd11 complex was also investigated for its ability to restore the growth deficiency of the E. coli 1iscS strain CL100 (**Figure 3C**). As expected, CL100 transformed with the plasmid containing the endogenous iscS gene restored the growth defect. Drosophila Nfs111–53/Isd11 partially complemented IscS function in the E. coli CL100 strain, whereas expression of IscU along with Nfs111–53/Isd11 slowed down bacterial growth (**Figure 3C**). Notably, this in vivo result matches the in vitro observation that L-cysteine binding to Nfs111–<sup>53</sup> is slower in the presence of Isd11 and IscU (**Figure 3B**). These findings are consistent with the previous demonstration that the L-cysteine desulfurase activity of the human NFS1/ISD11 complex is reduced in the presence of ISCU (Tsai and Barondeau, 2010; Bridwell-Rabb et al., 2014).

FIGURE 2 | Schematic comparison of Drosophila Nfs1 and human NFS1. Highly conserved residues that play a role for desulfurase catalysis and disease are indicated: the active site cysteine (C) that forms the persulfide intermediate (Zheng et al., 1994; Lauhon et al., 2004); the tyrosine (Y) was shown to be crucial for activity in Saccharomyces cerevisiae Nfs1p (Mühlenhoff et al., 2004); the point mutation p.Arg72Gln (arrow) in human NFS1 was identified to cause infantile mitochondrial complex II/III deficiency (Farhan et al., 2014); whereas histidine (H) and lyine (K) are involved in pyridoxal 5′ -phosphate (PLP) coordination (Cupp-Vickery et al., 2003). The proposed N-terminal mitochondrial targeting sequences is shown by a red box, while the nuclear localization signal is shown as a blue box. The beginning of the truncated Nfs1/NFS1 versions used for biochemical purification are indicated by an arrow head (Marelja, 2013). Numbers indicate amino acid position in the Homo sapiens (H.s.) and D. melanogaster (D.m.) proteins.

FIGURE 3 | In vitro characterization of Drosophila Nfs1, Isd11, IscU in complex with human frataxin. (A) Co-expression of Nfs111–<sup>53</sup> with Isd11 and/or IscU in Escherichia coli. After purification of the complexes with affinity chromatography, 30µM of Nfs111–<sup>53</sup> was applied onto the analytical Superdex200 size exclusion column and compared by SDS-PAGE. The fractions show that the Nfs111–53/Isd11 complex eluted earlier than the IscU/Nfs111–<sup>53</sup> complex, indicating its smaller size. As expected, the complex between all three proteins was larger. Human frataxin added to the purified Nfs111–53/Isd11/IscU complex also bound as demonstrated by further increase in size. Gamma globulin (158 kDa) and ovalbumin (44 kDa) were standards (kDa ladder on the top; fractions in ml units). (B) UV-Vis absorption spectra of 10µM Nfs111–<sup>53</sup> in complex with His6-Isd11 (top panel) and Isd11/His6-IscU (bottom panel) in the absence (solid black line) or 1 min and 5 min after addition of 4 mM L-cysteine (red lines). All spectra were recorded at 23◦C in 500 µl 100 mM Tris (pH 8.0), 200 mM NaCl, 1 mM EDTA. (C) Growth curves of 100 ml cultures of the Escherichia coli CL100 strain (1iscS) transformed either with vectors pET15b and pACYCDuet-1 or with plasmids containing coding sequences for Drosophila Nfs111–53, Isd11, IscU, or Escherichia coli IscS were recorded after addition of 100µM IPTG for 12h at 30◦C. (D) Desulfurase activity of Nfs111–53/Isd11 and Nfs111–53/Isd11/IscU complexes in absence or presence of human FXN. L-cysteine desulfurase activity was measured by determination of total sulfide produced. One unit is defined as the amount of enzyme producing 1 µmol of sulfide/min. Under anaerobic conditions, Nfs111–53/Isd11 and Nfs111–53/Isd11/IscU complexes were mixed in a 1:3 [Nfs1]:[FXN] ratio and incubated for 10 min at 23◦C. The last lane (hFXN +) is a negative control to show that the human FXN preparation does not carry contaminant desulfurase activity. Error bars indicate the standard deviation of three measurements.

To further test this notion, we tested whether IscU altered the desulfurase activity of purified Nfs1 in vitro (**Figure 3D**). Sulfide production activity was determined with methylene blue in the presence of 1,4 dithiothreitol (Fogo and Popowsky, 1949; Urbina et al., 2001). Only weak activity was detected from the purified Nfs111–53/Isd11 complex, possibly because the complex was unable to perform reaction turnovers. Addition of IscU, forming the IscU/Nfs111–53/Isd11 complex, abolished sulfide production, consistent with observations presented above. Addition of human frataxin to the ternary IscU/Nfs111–53/Isd11 complex led to a six-fold increase in sulfide production activity, showing that frataxin stimulated the desulfurase activity of Nfs1/Isd11 in an IscU-dependent manner (**Figure 3D**). Steady-state kinetic parameters were determined by varying L-cysteine concentration for Nfs111–<sup>53</sup> activity (after Nfs111–53/Isd11/IscU/frataxin complex formation) showing a catalytic efficiency of 149 M−<sup>1</sup> s −1 , kcat 2.6 min−<sup>1</sup> , and a K<sup>M</sup> of 0.290 mM. Taken together, these data indicate that Isd11, IscU, and frataxin are required for the activation of Nfs1 in Drosophila, providing experimental evidence that the initial complex of the mitochondrial Fe-S cluster biosynthetic machinery is conserved in Drosophila similar to what is known in other eukaryotes, including species with nonrespiring mitochondria (Tovar et al., 2003; Richards and van der Giezen, 2006).

#### Fe-S Cluster Biosynthesis Occurs in Distinct Cellular Compartments

Like the other proteins of the core complex described above, Drosophila Isd11 is localized in mitochondria (**Figure 4A**). Two mitochondrial ferredoxins (Palandri et al., 2015) and the Drosophila homolog of ferredoxin reductase (also known as adrenodoxin reductase encoded by the dare gene; Freeman et al., 1999) are required as electron donors for Fe-S cluster formation (Cai et al., 2017). Mitochondrial monothiol glutaredoxin-5 (CG14407) has not been investigated in Drosophila, but is likely required for Fe-S cluster biosynthesis (Rodríguez-Manzaneque et al., 2002; Wingert et al., 2005; Ye et al., 2010; Johansson et al., 2011), participating in the Fe-S cluster transfer to BolAlike proteins (Aldea et al., 1988; Frey et al., 2016; Melber et al., 2016; Uzarska et al., 2016; Nasta et al., 2017). Fe-S clusters are also transferred to Nfu-like (Tong et al., 2003; Melber et al., 2016; Wachnowsky et al., 2016) or Isa-like proteins (Jensen and

FIGURE 4 | Subcellular localization of Isd11 and Mocs3. Confocal microscopy of (A) Hela cells transfected with a C-terminal fusion of EYFP to Isd11, stained with Mitotracker Red to detect mitochondria and with DAPI to visualize nuclei. Insets show two channels separately to appreciate co-localization of the fluorescent signals from Isd11-EYFP and Mitotracker (merged, bottom inset); (B) Hela cells transfected with Mocs3-EYFP, which localizes in the cytosol and nuclei.

Culotta, 2000; Kaut et al., 2000; Muhlenhoff et al., 2011; Sheftel et al., 2012) with the chaperone activity of mitochondrial Hsc20 (Uhrigshardt et al., 2010; Sieber et al., 2016) and its cognate Hsc70 [not clear which of several candidate Hsc70 genes present in the fly genome (Adams et al., 2000) functions in Fe-S cluster biosynthesis]. These scaffold proteins deliver the Fe-S clusters to target mitochondrial Fe-S enzymes.

Fe-S clusters are also assembled on proteins in the cytosol (**Figure 1**). Early work in this area suggested that core mitochondrial Fe-S cluster assembly proteins were also being directed to the cytosol (Land and Rouault, 1998; Tong and Rouault, 2000, 2006) and protein targeting sequences do not typically result in a unique destination for most proteins (Hegde and Bernstein, 2006). Furthermore, another set of cytosolic Fe-S cluster assembly (CIA) proteins has been described (reviewed in Roy et al., 2003; Hausmann et al., 2005; Paul and Lill, 2015). Briefly, an Fe-S cluster is assembled on CFD1 and NBP35 with electron donors provided by CIAPIN1. The clusters are transferred through IOP1 to a scaffold with CIA1, CIA2, and target proteins. These CIA proteins are conserved in Drosophila (**Figure 1**), but little work exists in the context of Fe-S cluster assembly. The fly Ciao1 homolog was shown to be required for viability (Radford et al., 2005) prior to assigning its biochemical function as part of the Fe-S scaffold complex in CIA (Balk et al., 2005a). Ciao1 received its name from the word "bridge" in the Chinese language (Johnstone et al., 1998), whereas the yeast homolog was conveniently designated Cia1 when its interaction with the hydrogenase-like Nar1 (IOP1 in the human nomenclature; Huang et al., 2007) was discovered (Balk et al., 2005b).  The Drosophila CIAPIN1 homolog is required for oogenesis (Marzuk et al., 2013). CIAPIN1 carries an Fe-S cluster (Zhang et al., 2008) and receives electrons from a cytosolic reductase (Netz et al., 2010). Neither the reductase nor cytosolic monothiol glutaredoxins (Li et al., 2009; Mühlenhoff et al., 2010; Banci et al., 2015) have been characterized in Drosophila. Furthermore, it is important to stress that no experimental data exist to implicate the Drosophila proteins Cfd1, Ciao1, CIAPIN1, Galla-1, Galla-2, Iop1 Mms19, Nbp35 in the assembly of cytosolic Fe-S clusters; there inclusion here and elsewhere (Mandilaras and Missirlis, 2012) as members of the CIA was exclusively based on gene homology searches. Nevertheless, many projects are being undertaken in other systems to better describe the process of Fe-S cluster assembly in cytosol and nucleus and we hope to see contributions from studies in Drosophila in the near future.

Finally, it is noteworthy that no Fe-S enzymes have been reported in the secretory pathway, implying that the presence of thiol-reducing activity in the cytosol, nucleus and mitochondrial matrix is essential for their stability. In insects, such activity rests on the thioredoxin reductase system (Kanzok et al., 2001; Missirlis et al., 2001, 2002).

#### Excess Copper Inhibits Fe-S Cluster Biosynthesis

Copper and iron homeostasis are intimately linked (Fox, 2003). Physiologically, the two metals are required for aerobic respiration, albeit as separate cofactors (Villee, 1948). Poulson and Bowen made an early observation that when Drosophila larvae were fed a diet rich in copper their iron stores were being depleted (Poulson and Bowen, 1952). A key way in which the two metals depend on each other was uncovered when ceruloplasmin and other multicopper oxidases (MCOs) were shown to act as ferroxidases (Osaki et al., 1966) and their activity was linked to iron trafficking across membranes (reviewed in Kosman, 2010). Drosophila MCOs are linked to iron homeostasis in ways that are still not understood (Bettedi et al., 2011; Lang et al., 2012; Peng et al., 2015), but MCO3 mutants fed on copper also show a reduction in ferritin iron content (Bettedi et al., 2011). It was therefore important to identify another hypothesis to explain copper-mediated cellular iron deficiency. The discovery that excess copper displaces the Fe-S cluster of mitochondrial ferredoxin (Vallières et al., 2017), leading to disrupted CIA (Alhebshi et al., 2012), suggests that copper toxicity may directly inhibit Fe-S cluster biosynthesis also in Drosophila. Reducing Fe-S cluster biosynthesis in Drosophila with either RNA interference (RNAi) of frataxin or loss-of-function Hsc20 mutants lead to mitochondrial iron accumulation and reduced ferritin expression (Anderson et al., 2005; Uhrigshardt et al., 2013; Navarro et al., 2015), offering a testable potential explanation of why excess dietary copper affects ferritin iron accumulation. In this respect, we also note that copper chelation ameliorated a fly model of Friedreich's ataxia (Soriano et al., 2016) and that the dithiol Drosophila glutaredoxin-1 was implicated in copper homeostasis (Mercer and Burke, 2016).

#### A Mitochondrial Iron Sulfur Enzyme Initiates Moco Biosynthesis

Turning to Moco biosynthesis, an ancient, ubiquitous and highly conserved pathway underpinning molybdenum biochemistry (Rajagopalan, 1997; Mendel and Leimkühler, 2015; Leimkühler, 2017), it can be divided into three major steps: (i) GTP is converted to cPMP, (ii) cPMP is converted to MPT by generation of the dithiolene group, and (iii) molybdate is then ligated to MPT forming Moco (**Figure 1**). Three fly genes involved in Moco biosynthesis have attracted individual attention: (i) Mocs1 (formerly known as low xanthine dehydrogenase, lxd) encoding a mitochondrial Fe-S enzyme that converts 5′ -guanosine triphosphate (GTP) to cyclic pyranopterin monophosphate (cPMP) (Keller and Glassman, 1964; Courtright, 1975; Duke et al., 1975; Bogaart and Bernini, 1981; Schott et al., 1986; Ho et al., 1992; Tahoe et al., 2002), (ii) cinnamon (cin) encoding a gephyrin homolog that inserts molybdate into MPT (Baker, 1973; Browder and Williamson, 1976; Kamdar et al., 1994; Wittle et al., 1999), and (iii) maroon-like (mal) that encodes a Moco sulfurase (Mitchell and Glassman, 1959; Hubby and Forrest, 1960; Finnerty et al., 1970; Marsh and Wieschaus, 1977; Kamleh et al., 2009).

Moco biosynthesis starts within mitochondria with a complex rearrangement reaction in which the C8 atom of the GTP purine is inserted between the 2′ and 3′ -ribose carbon atoms (Wuebbens and Rajagopalan, 1993; Hover and Yokoyama, 2015). The human MOCS1 gene is orthologous to the lxd locus (now renamed Mocs1) of D. melanogaster (Gray and Nicholls, 2000). Mutations in lxd affect molybdoenzyme activity in flies (Keller and Glassman, 1964). Alternative splicing at the Mocs1 locus results in short (Mocs1A) and full length (Mocs1A-Mocs1B) proteins (**Figure 5A**). Mocs1A belongs to the superfamily of SAM-dependent radical enzymes (Hänzelmann and Schindelin, 2004),requiring a [4Fe-4S] cluster for the formation of a substrate radical by reductive cleavage of SAM. Studies on the human and bacterial homologs showed that Mocs1B participates in pyrophosphate cleavage after the formation of the 3′ , 8cH2GTP

FIGURE 5 | Transcriptional units and proteins encoded by Drosophila genes involved in Moco biosynthesis. Transcript names are shown as per Flybase. Protein domains in light orange boxes are according to the nomenclature in bacteria (Leimkühler, 2017). Names of the Drosophila proteins as used in text are shown below each transcript. (A) Alternative splicing of Mocs1 results in short (Mocs1A) and long (Mocs1A-Mocs1B) subunits, which complex together to form the active enzyme. (B) A bicistronic transcript results in Mocs2B (which also carries the unrelated to Moco biosynthesis MBIP domain, see text) and Mocs2A. (C) Mocs3 carries the MoeB-like domain and a rhodanese-like domain (RLD), shown to interact with Nfs1 (Marelja et al., 2013). (D) Cinnamon carries the MogA/Gephyrin G domain at the N-terminus prior to the C-terminal MoeA/Gephyrin E domain (in reverse order to the mammalian Gephyrins).

intermediate (Hänzelmann and Schindelin, 2006). Evidence was sought for Mocs1 having a role in lifespan determination of Drosophila, as polymorphisms in the gene were detected between short- and long-lived inbred lines, but the results were inconclusive as the polymorphisms could not be associated with clear effects on enzyme activity (Tahoe et al., 2002). Mutants in Mocs1 showed differential sensitivity to dietary molybdate compared to wild type strains (Duke et al., 1975).

## Molybdenum Cofactor Biosynthesis in the Cytosol

In the second step, two sulfur atoms are transferred to cPMP to form MPT. This reaction is catalyzed by the heterotetrameric MPT synthase, which is composed of two small Mocs2A and two large Mocs2B subunits encoded from a single locus (Stallmeyer et al., 1999; Leimkuhler et al., 2003; **Figure 5B**). The physiological sulfur donor for MPT synthesis is Mocs3 (Matthies et al., 2004, 2005), which resides in the cytosol (**Figure 4B**). Drosophila Mocs2 and Mocs3 are both required for Aox activity (**Figure 6**). Based on what is known in human cells (Marelja et al., 2008), Mocs3 is expected to receive sulfur from Nfs1, the same protein that acts as a sulfur donor for Fe-S cluster biosynthesis in mitochondria, to which it binds through a rhodanese-like domain (RLD; **Figure 5C**). Using Förster resonance energy transfer and a split-EGFP system, NFS1 was shown to interact in the cytosol of human cells with MOCS3 (Marelja et al., 2013). This result was corroborated by showing that human NFS1/ISD11 requires MOCS3 to complement Moco biosynthesis in the E. coli deletion strain used in **Figure 3C** (Bühning et al., 2017).

Exceptionally, the Drosophila MPT synthase is linked to c-Jun N-terminal kinase (JNK) signaling, since Mocs2B forms a fusion protein with the mitogen activated protein kinase upstream binding inhibitory protein (MBIP; **Figure 5B**), which is one of 13 subunits of the Ada Two A containing histone acetyltransferase complex (ATAC) transcriptional co-activator (Suganuma et al., 2010). In human cells, Mocs2B/MBIP were shown to regulate ferritin translation through inhibition of PKR, a double-stranded RNA-dependent protein kinase (Suganuma et al., 2012, 2016). Whether this form of regulating iron storage is conserved in Drosophila, whether it serves to regulate the availability of Fe-S clusters for Mocs1, and whether Drosophila Nfs1 has a cytosolic function are questions for future research.

In the last step, molybdate is ligated to the dithiolene group of MPT producing Moco (**Figure 1**). The first results for the enzyme inserting molybdate to the pterin structure came from the Xdh deficiency of the cin mutation in Drosophila (Baker, 1973). Cin is partially homologous to the two E. coli proteins MogA and MoeA, which are also found as domains G and E of the rat protein Gephyrin, albeit in reverse orientation to the Drosophila protein (Kamdar et al., 1994; Stallmeyer et al., 1995; Feng et al., 1998; **Figure 5D**). The MogA-like G domain binds MPT and catalyzes the MPT-adenylation from Mg-ATP; MPT-AMP is then transferred to the MoeA-like E domain for hydrolysis and molybdenum insertion (Kuper et al., 2000, 2004; Schwarz et al., 2000; Llamas et al., 2004, 2006). Furthermore, the central domain in Gephyrin binds and anchors inhibitory

FIGURE 6 | RNAi of Moco biosynthesis genes disrupts Aox activity. In-gel Aox activity is shown as described before (Marelja et al., 2014). Extracts from wild type flies are shown in column 1 of the gels, followed by extracts from RNAi of, respectively, Mocs2, Mocs3, cinnamon, and maroon-like in column 2 (note reduced Aox activity, consistent with a requirement of these genes for Moco biosynthesis). Columns 3 and 4 are extracts from the parental flies of the Gal4/UAS system that show normal Aox activity (controls), as do extracts from white and rosy mutants in columns 5 and 7 (positive controls), whereas extracts from the mal mutant show no detectable Aox activity (negative control, column 6).

ligand-gated anion channels in the postsynaptic membrane of neurons (Feng et al., 1998; Stallmeyer et al., 1999). However, it is unknown whether this additional function is conserved for Drosophila Cin.

After molybdenum insertion into MPT, Moco is either inserted into Suox or further modified by exchanging an oxo ligand by a sulfido group (Hille, 1996, 2002; Hille et al., 2014). The sulfur incorporation was also first discovered in the fly mal mutant, which lacked Xdh and Aox activities (Hadorn and Mitchell, 1951; Glassman and Mitchell, 1959; Hubby and Forrest, 1960; Forrest et al., 1961; Courtright, 1967) but retained or induced Suox activity (**Figure 7**) and an apparently normal concentration of total Moco (Bogaart and Bernini, 1981; Warner and Finnerty, 1981; Wahl et al., 1982). In vitro reconstitution of Xdh and Aox activities with sulfide/dithionite treatment led to the suggestion that mal affected the sulfur modification after the Mo insertion (Wahl and Rajagopalan, 1982; Wahl et al., 1982). A sequence comparison with the L-cysteine desulfurase gene nifS from Azotobacter vinelandii suggested that the mal gene encodes the enzyme that catalyzes sulfuration of Moco used by Xdh and Aox (Amrani et al., 2000; Ichida et al., 2001). Oxo-containing Moco does not function in Xdh and Aox, but is the cofactor for Suox (**Figure 1**).

# Fe-S AND MOCO ENZYMES

The fly offers an ideal system for interdisciplinary research to bridge findings from genetic manipulations and resulting phenotypes with detailed biochemical studies to build functional understanding for animal cell physiology, keeping in mind the diversity of cell types and subcellular microenvironments.

In this section, we review the enzymes that use Moco and/or Fe-S clusters. A representative list of these enzymes in D. melanogaster is provided (**Table 1**). We emphasize the physiological functions and biochemical pathways, as opposed to the detailed biochemistry of the cofactors at the active sites, which only in few cases has been the primary interest of Drosophila researchers.

# The Molybdoenzymes: Xdh, Aox, Suox

In D. melanogaster, the rosy gene encodes Xdh (Chovnick et al., 1976; Keith et al., 1987). Rosy mutants accumulate xanthine and hypoxanthine and are devoid of urate (Hadorn and Schwinck, 1956; Morita, 1958; Glassman and Mitchell, 1959) and show a dull reddish-brown eye color because of the lowered concentrations of the red eye pigment drosopterin (bright red), and the increased chromogenic oxidation of the eye pigment dihydroxanthommatin (yellow brown) to xanthommatin (dull dark brown), either due to enzymatic activity (Phillips and Forrest, 1980; Wiley and Forrest, 1981; Ferre et al., 1986) or due to the decreased urate concentration (Hilliker et al., 1992). The use of rosy mutant strains to probe the structure and function of Xdh provides an early, elegant example in the field of biological inorganic chemistry (Hughes, 1992; Hughes et al., 1992; Doyle et al., 1996). Allelic series of mutants affecting residues coordinating the Fe-S clusters, the Moco, the flavin cofactor or the binding sites for NAD+/NADH were used to decipher the complex mechanism of action for this prototypical molybdoenzyme. More recently, metabolic profiling of rosy mutants revealed additional changes in the tryptophan, arginine, pyrimidine, and glycerophospholipid metabolic pathways (Kamleh et al., 2008, 2009).

Two genes, low pyridoxal oxidase (lpo) and aldehyde oxidase (Aldox), were initially recognized to encode Aoxs in D. melanogaster (Courtright, 1967; Collins and Glassman, 1969; Dickinson, 1970; Browder and Williamson, 1976; Dickinson and Gaughan, 1981; Cypher et al., 1982; Nelson and Szauter, 1992). Genome analysis revealed, however, a cluster of four Aox genes (Garattini et al., 2008). We showed previously that (i) the lpo gene encodes Aox1, (ii) Aox2 activity is only present during metamorphosis, (iii) the activities associated with the classic Aldox locus correspond to two splice forms of the Aox3 gene, and (iv) no major activity has been found associated with Aox4, the newest addition to a highly evolving protein family in Drosophilidae (Marelja et al., 2014). Aoxs show specific substrate specificities, but their in vivo substrates and physiological functions remain unclear (Dickinson and Gaughan, 1981; Cypher et al., 1982; Marelja et al., 2014).

Suox contains a cytochrome b5 (heme-containing) domain followed by 12–15 amino acids connecting it to the Moco domain. Suox catalyzes an oxygen atom transfer reaction to sulfite leading to its oxidation (and detoxification) to sulfate (Feng et al., 2007; Hille et al., 2011), an activity previously detected in Drosophila (Bogaart and Bernini, 1981; Braaten and Bentley, 1993). Based on sequence homology, we find that CG7280 encodes for the Drosophila Suox.

A novel molybdoenzyme, mitochondrial amidoxime reducing component (mArc), has been described and has an identifiable Drosophila homolog, CG1665. The enzyme from humans has been proposed to reduce a broad range of Nhydroxylated compounds receiving electrons from cytochrome b5 (Gruenewald et al., 2008) and to reduce nitrite to nitric oxide (Sparacino-Watkins et al., 2014). A number of controversies over the function, subcellular localization and whether the newly discovered enzyme binds to sulfurated or oxo Moco, have been reviewed (Llamas et al., 2017).

Another relatively unexplored area in Drosophila molybdenum biology relates to the trafficking and homeostatic mechanisms for handling dietary molybdate. In wild type flies, Xdh and Aox activities are relatively stable, not responding to concentrations between 1 and 10 mM molybdate (Duke et al., 1975), although more recent studies documented a number of, so far unexplained, sex-specific physiological responses in carbohydrate and thiol metabolism at the same concentrations (Rovenko et al., 2014; Perkhulyn et al., 2017). Interestingly, 50 mM molybdate was tolerated by wild type flies, but was TABLE 1 | List of known molybdoenzymes, iron sulfur enzymes and scaffold proteins.

lethal to Mocs1 mutant flies, implicating the Moco biosynthetic pathway as part of the detoxification mechanisms available to the fly (Duke et al., 1975).

#### The Curious Case of the Maroon Mutant

A second unusual aspect of Xdh, besides its implication in multiple metabolic pathways (see above Kamleh et al., 2008, 2009), relates to the enzyme's trafficking to the eye imaginal disk, where it accumulates in pigment granules (Reaume et al., 1989, 1991). Due to the similar eye color of the classic maroon mutant with that of the rosy and mal mutants (Bridges, 1918), we assayed maroon fly extracts for Xdh and Suox activity, expecting to find decreased Xdh activity consistent with the eye phenotype. To our surprise, increased activities of both enzymes compared to wild type flies were observed in maroon extracts (**Figure 7**). As Suox utilizes unmodified Moco, the increase in its activity was not entirely unexpected, because this form of Moco might accumulate as in rosy and mal. Accounting for increased Xdh activity was harder, however, given the similar eye color between maroon, rosy, and mal. The demonstration that the maroon gene encodes for Vacuolar protein sorting 16A (Vps16A; Grant et al., 2016), a protein implicated in granule formation (Pulipparacharuvil et al., 2005; Lorincz et al., 2016), suggests a possible defect in the tissue localization of Xdh in maroon mutants, as the enzyme obviously remains functional in whole fly extracts and is even induced (**Figure 7**). It will be informative to test in maroon mutants whether the Xdh activity is localized in peripheral tissues, such as the Malpighian tubules, and not in the eyes. If this prediction is correct, then Vps16A may represent the first known mutant that blocks the delivery of a vesicular structure to the eye. Another unresolved piece of this puzzle relates to the way in which Xdh, a cytosolic enzyme, is found in pigment granules in the eye (Reaume et al., 1989, 1991). The discovery of exosomes gives a possible answer to this conundrum (Hemler, 2003; Gross et al., 2012; Gradilla et al., 2014; Takeuchi et al., 2015; Beer and Wehman, 2017; Shibata et al., 2017; Tassetto et al., 2017). Clearly more experiments are required to explain how Xdh acts in the formation of eye color in flies, but complex non-cell autonomous processes relating to enzyme maturation, regulation, and transport are involved.

# Mitochondrial Fe-S Proteins at the Heart of Mitochondrial Bioenergetics

Mitochondria form an important organelle of eukaryotic cells, typically containing their proper genome (Lane and Martin, 2010; Schatz, 2013; Allen, 2015) and performing various functions (Pagliarini and Rutter, 2013; Chandel, 2015; Munro and Treberg, 2017), of which the tricarboxylic acid (TCA) cycle and oxidative phosphorylation are famous (Vakifahmetoglu-Norberg et al., 2017). The idea that iron plays a part in the oxidation reactions of the living cell was firmly established by Warburg (1925). Beinert and Sands interpreted electron paramagnetic resonance spectra of succinate dehydrogenase (Sdh) as "non heme iron" (Beinert and Sands, 1960; Beinert, 2002). Later, Sdh and aconitase (both TCA cycle enzymes) were shown to carry [4Fe-4S] clusters (Ruzicka and Beinert, 1978; Cammack, 1982).  Failure to build these Fe-S clusters will inevitably block the TCA cycle, and thereby development and growth (Au and Scheffler, 1994; Yan et al., 1997; Walker et al., 2006; Uhrigshardt et al., 2013; Na et al., 2014). Sdh is also known as Complex II of the respiratory chain that generates the inner mitochondrial membrane potential and proton gradient used by the F-ATPase for the production of ATP during oxidative phosphorylation (Alziari et al., 1985; Sardiello et al., 2005; Liu et al., 2011; Barry and Thummel, 2016). Complex I of the respiratory chain, otherwise known as NADH dehydrogenase, carries eight precisely spaced Fe-S clusters of different reduction-oxidation potentials assembled on five subunits of the complex (**Table 1**; Zhang K. et al., 2013; Fiedorczuk et al., 2016; Garcia et al., 2017). It is thus plain that without Fe-S clusters ATP cannot be produced in the mitochondrial process of aerobic respiration (Anderson et al., 2005; Llorens et al., 2007; Navarro et al., 2011; Edenharter et al., 2017).

### Fe-S Enzymes Are Required for Heme, Ecdysone, and Lipoate Biosynthesis

Ferrochelatase is an enzyme that resides in the inner mitochondrial membrane accepting iron from the mitochondrial matrix and protoporphyrin IX from the intermembrane space to generate heme (Wu et al., 2001). Heme is another abundant iron-dependent protein cofactor (Ponka et al., 2017). Many ferrochelatases, including the Drosophila enzyme, carry a [2Fe-2S] cluster (Sellers et al., 1998). Therefore, both major forms of iron cofactors used in biology rest on the mitochondrial Fe-S cluster assembly machinery.

Furthermore, Drosophila ferredoxins carry a [2Fe-2S] cluster required for electron transfer during Fe-S cluster assembly, but also for the production of ecdysone in the larval prothoracic gland and other steroidogenic tissues (Uhrigshardt et al., 2013; Palandri et al., 2015). In this way, iron availability is linked to a key developmental signal that terminates growth and initiates metamorphosis (Yamanaka et al., 2013; Sandoval et al., 2014). Interestingly, glutathione production in the prothoracic gland is also required for steroidogenesis (Enya et al., 2017). The possibility that glutathione supports Fe-S cluster biosynthesis in this tissue should be considered (Song et al., 2006; Auchère et al., 2008; Qi et al., 2013; Ozer et al., 2015).

Lipoic acid or lipoate is a cofactor required in intermediary metabolism enzymes α-oxoglutarate dehydrogenase, pyruvate dehydrogenase, branched-chain oxoacid dehydrogenase, 2 oxoadipate dehydrogenase, and in the glycine cleavage system (Habarou et al., 2017). Biosynthesis of the lipoate cofactor is not well understood beyond prokaryotes (for comprehensive review see Cronan, 2016). Nevertheless, it is clear that lipoic acid synthase is required for the maturation of enzymes dependent on lipoate and uses two [4Fe-4S] clusters for its catalytic activity (Harmer et al., 2014). No study describing the Drosophila lipoic acid synthase has been published despite human disease related to lipoic acid deficiency (Mayr et al., 2014; Cronan, 2016; Habarou et al., 2017).

# The Central Dogma of Molecular Biology Depends on Fe-S Enzymes

The central dogma of molecular biology, originally proposed by Crick (1958), radically changed the way biology is understood and taught (Cobb, 2017). DNA replication is the primary mode of information transfer during successive generations, whereas DNA transcription is the primary mechanism for specifying which proteins can be translated on ribosomes assembled in the cytoplasm. In addition to the nucleotide and amino acid building blocks, all three steps require energy and are interdependent, as nucleic acids and proteins are both essential for each process. Likewise, Fe-S clusters are also required at each step as cofactors of the DNA polymerase (see references in **Table 1** and Kaguni et al., 1983; Peck et al., 1992; Sahashi et al., 2014; Stiban et al., 2014), of the essential subunit of the basal transcription factor TFIIH Xpd (Reynaud et al., 1999; Chen et al., 2003; Aguilar-Fuentes et al., 2006; Li et al., 2010) and of Pixie, which is required for ribosome biosynthesis and the initiation of translation (Andersen and Leevers, 2007; Kashima et al., 2014). Thus, DNA replication, transcription, and translation rest on the CIA providing Fe-S cluster to DNA polymerase, Xpd, and Pixie, respectively.

## Are Fe-S and/or Moco Enzymes Implicated in the Circadian Clock?

Plants alternate between photosynthesis and respiration during day-night cycles, whereas animal behavior shifts between an active stage that includes foraging, feeding and other motile behaviors, and sleep (Haydon et al., 2013; Mellor, 2016; Dubowy and Sehgal, 2017). Most animals anticipate the periodicity of sunlight and darkness through dedicated neuronal circuits whose rhythmic activity is sometimes referred to as the circadian clock (the genetic basis of which, was first discovered by Konopka and Benzer, 1971). In Drosophila the organization of the circadian circuitry has received considerable attention (Nitabach and Taghert, 2008; Hermann et al., 2013; Simoni et al., 2014). Similar to other animals, the circadian clock is interlinked with physiological functions in flies (Barber et al., 2016; Katewa et al., 2016; Rey et al., 2016; Kijak and Pyza, 2017; Klemz et al., 2017). Given the major role of Fe-S enzymes in intermediary metabolism and aerobic respiration, the question of whether Fe-S clusters are continuously present in key enzymes during the daynight cycle or whether some recycling of iron takes place in a rhythmic function has been posed (Mandilaras, 2012).

RNAi of Nfs1 (the cysteine desulfurase required for the biosynthesis of Fe-S clusters and Moco; **Figure 1**) in the circadian clock neurons resulted in loss of rhythmic activity of flies monitored under constant darkness (Mandilaras and Missirlis, 2012). Ubiquitous RNAi of Nfs1 caused lethality and eye-specific RNAi caused photoreceptor cell loss (Marelja, 2013). Two driver lines with overlapping, but not identical, expression patterns in the clock neurons, tim27-Gal4 and cry17<sup>b</sup> -Gal4 were recombined to the RNAi potentiator UAS-Dicer2 (Dietzl et al., 2007) and used, showing that Nfs1 RNAi driven by tim27-Gal4 resulted in a weaker arrhythmia than when driven by cry17<sup>b</sup> - Gal4 (Mandilaras and Missirlis, 2012). IscU RNAi driven by cry17<sup>b</sup> -Gal4 also resulted in arrhythmic flies, IscU RNAi driven by tim27-Gal4, however, resulted in lethality (a more severe phenotype, but one that cannot be tested for rhythmicity). In contrast, frataxin RNAi with both drivers did not show an arrhythmic phenotype (Mandilaras and Missirlis, 2012). To probe these genetic results further, RNAi of Isd11 and Mocs3 using the same drivers and assay was undertaken. Isd11 RNAi in circadian clock neurons showed no discernible phenotype, in contrast to Mocs3 RNAi, which resulted in 56% arrhythmic flies when driven with tim27-Gal4 and 64% arrhythmic flies when driven with cry17<sup>b</sup> -Gal4 (**Figure 8**).

The other Fe-S cluster biosynthesis genes that were implicated in the Drosophila circadian clock were Iba57 (CG8043) and Cfd1 (CG4858; also referred to as Nubp2), both encoding components of the CIA machinery, and IscA1, which is an Fe-S scaffold protein predicted to be localized to mitochondria (Mandilaras and Missirlis, 2012). IscA1 has recently found an exciting application in a new, non-invasive, technology to control experimentally the generation of neuronal action potentials, termed magnetogenetics (Long et al., 2015). It was also suggested that IscA1 is a component of fly magnetoreception (Cyranoski, 2015; Qin et al., 2016), but see (Meister, 2016; Pang et al., 2017). The proposed cryptochrome-IscA1 protein complex is predicted to reside in the cytosol or nucleus, given that cryptochrome is a protein localized in the cytosol and nucleus (Stanewsky et al., 1998; Yoshii et al., 2008). Whether, in addition to their predominant presence in mitochondria, IscA1, IscU, and Nfs1 have cytosolic localization in flies, as recently shown for human HSC20 (Kim et al., 2018), requires experimental verification. It is an interesting possibility to keep in mind when considering the result that RNAi against Mocs3, whose product is the proposed cytosolic partner of Nfs1 (Marelja et al., 2008, 2013), resulted in arrhythmic flies (**Figure 8**). Of note, two Fe-S enzyme encoding genes, dihydropyrimidine dehydrogenase and phosphoribosylamidotransferase, show circadian expression and are localized in the cytosol (Van Gelder et al., 1995; Rey et al., in review).

The new results presented here and the findings in Mandilaras and Missirlis (2012) are only based on RNAi and require independent confirmation. That iron may play a role in the circadian clock and sleep disorders was also proposed based on a Drosophila model of the human Restless Legs Syndrome (Freeman et al., 2012, 2013).

# Cellular Iron Sensing and Regulation Is Coupled to Fe-S Cluster Biosynthesis

D. melanogaster larvae or flies grown on diets with different iron content show cell-type specific responses (Poulson and Bowen, 1952; Georgieva et al., 1999; Mehta et al., 2009; Mandilaras et al., 2013). In mammals, IRPs regulate cytosolic

values are provided for flies scored as rhythmic: higher values demonstrate more robust rhythmicity (for detailed methodology see Mandilaras and Missirlis, 2012).

iron concentrations (Zhang et al., 2014; Muckenthaler et al., 2017; Papanikolaou and Pantopoulos, 2017; Rouault and Maio, 2017). D. melanogaster has two IRP-like proteins, showing a partial evolutionary conservation in its iron sensing mechanism (Muckenthaler et al., 1998; Lind et al., 2006; Freeman et al., 2013). Another conserved aspect in cellular iron homeostasis is that genetic manipulations that affect mitochondrial Fe-S cluster biosynthesis lead to cytosolic iron depletion (Anderson et al., 2005; Uhrigshardt et al., 2013; Navarro et al., 2015). The mechanism of this response is not fully conserved between yeast (Wofford and Lindahl, 2015) and vertebrates (Wingert et al., 2005; Ye et al., 2010), as the former lack IRPs, and it is not known how Drosophila cytosolic iron is regulated through the mitochondrial Fe-S cluster assembly machinery. Furthermore, transcriptional regulation of ferritin upon iron sensing appears to dominate the Drosophila iron response (Georgieva et al., 1999; Missirlis et al., 2007; Rosas-Arellano et al., 2016), suggesting that an undiscovered transcription factor responds to varying cytosolic iron concentration in insects. Of note, Drosophila ferritin is loaded with iron in the endoplasmic reticulum (Xiao et al., 2014; Xiao and Zhou, 2018) and is an essential gene (González-Morales et al., 2015), as is also the case for IRP-1A (Puri et al., 2008). More work is needed to understand cellular iron homeostasis in Drosophila.

### How Do Mitochondria Regulate Their Iron Needs?

Mitochondria use a specialized ferritin (Missirlis et al., 2006) and at least one specialized transporter for iron import into the mitochondrial matrix, mitoferrin (Metzendorf and Lind, 2010; Navarro et al., 2015; Edenharter et al., 2017). The question how they sense and regulate iron concentration in the matrix is not resolved. One unexplained observation in this respect is that, at least in yeast, the GTP to GDP ratio affects iron concentration within this compartment (Gordon et al., 2006). In what follows, we explore the idea that manganese Sod2 (Kirby et al., 2002; Duttaroy et al., 2003) may serve as a mitochondrial iron sensor (**Figure 9**). Our view of normally respiring mitochondria is the familiar setting with an active Sod2 protecting Fe-S clusters and maintaining the TCA cycle (**Figure 9A**; Missirlis et al., 2003a). An increase in superoxide (Wong H. S. et al., 2017) deactivates the Fe-S clusters in aconitase and Sdh (Gardner and Fridovich, 1991; Gardner et al., 1995) leading to an increase in ferrous iron in the mitochondrial matrix (Srinivasan et al., 2000; Jensen et al., 2004; Esposito et al., 2013). The literature on these reactions has been reviewed with a discussion of the accompanying consequences for cell metabolism (see superoxide/aconitase rheostat model; Armstrong et al., 2004). Increased mitochondrial iron can replace manganese on Sod2 and inactivate the enzyme (Yang et al., 2006; Naranuntarat et al., 2009). Sod2 inactivation would result in a positive feedback loop, as more superoxide would accumulate, fully inactivating aconitase and Sdh bringing the TCA cycle to a halt.

Heping Cheng and co-workers described a superoxide burst in mitochondria, termed mitoflash, lasting for several seconds (Wang et al., 2008; Shen et al., 2014). Some skepticism on whether the phenomenon is real exists, mostly arguing that perhaps the change in the fluorescence of the reporter used relates to pH changes (Schwarzländer et al., 2012, 2014), but the coupling of TCA cycle with oxidative phosphorylation could mean that both claims (a burst in superoxide and alkalinization of the mitochondrial matrix) occur at similar timeframes or in parallel (Wei-LaPierre et al., 2013).

During the inactivation of Sod2, citrate cannot be isomerized to isocitrate and is therefore exported to the cytosol for fatty acid synthesis (see below). Likewise succinate cannot be metabolized, but its precursor, succinyl-CoA is a substrate of aminolevulinate synthase (Alas) in the first step of heme biosynthesis (Ruiz de Mena et al., 1999; Ponka et al., 2017). Thus the superoxide burst couples the redirection of iron and intermediary metabolites to ferrochelatase for heme production (**Figure 9B**). This way, iron concentration in the mitochondrial matrix drops, manganese binds to and reactivates Sod2, and the mitochondria return to their conventional state. In the above discussion we have not considered the role of hydrogen peroxide, produced by Sod2, which should be treated as a distinct metabolite (Missirlis et al., 2003b; Munro and Treberg, 2017).

FIGURE 9 | A model showing two different states of mitochondrial metabolism. (A) Conventional state based on the TCA (Krebs) cycle feeding reducing equivalents into the respiratory chain for the production of ATP. Only metabolites and enzymes referred to in the text are shown for simplicity. (B) A superoxide burst is predicted to reduce the [4Fe-4S] clusters of aconitase and Sdh, releasing ferrous iron into the mitochondrial matrix and blocking the TCA cycle. The resulting increase in iron concentration may inactivate manganese Sod2 resulting in a sustained pulse of superoxide, previously referred to as a "mitoflash." In this state, mitochondria export citrate to the cytosol for fatty acid production, Alas uses up succinyl-CoA for protoporphyrin biosynthesis, ferrochelatase uses protoporphyrin and iron to produce heme. The consequent drop in matrix soluble iron reactivates manganese Sod2, allowing for Fe-S cluster biosynthesis to reactivate the TCA cycle enzymes.

Although the metabolites that are affected in the Sod2 mutants remain to be described, a recent paper reported a four-fold induction of aminolevulinic acid in Sod1 mutants, and curiously, a 20-fold induction of citrulline (Doran et al., 2017). Citrulline production depends on (the heme-containing) nitric oxide synthase (Nos), which uses as substrate arginine (Kuzin et al., 1996; Jaszczak et al., 2015). Superoxide-dependent inactivation of another manganese-containing enzyme, arginase, which degrades arginine (Samson, 2000), could lead to increased substrate availability for Nos. Indeed, it has been shown that arginase inactivation induces Nos activity (Caldwell et al., 2015). The above suggest another testable example of a similar hypothesis, where the interaction of superoxide with Fe-S clusters influences cell metabolism by releasing iron, which could transiently displace manganese from the active site of its target enzyme(s).

#### Fe-S Clusters and Lipid Metabolism

Citrate is a key precursor for fatty acid synthesis (Watson and Lowenstein, 1970; Halperin et al., 1975) and therefore it is not surprising that inactivation of aconitase leads to increased lipogenesis (reviewed in Tong and Rouault, 2007). The same metabolic connection has also been demonstrated in Drosophila, by driving frataxin RNAi in glial cells (Navarro et al., 2010) or in photoreceptor mutant clones (Chen et al., 2016b). Another outcome of increased mitochondrial iron due to loss of frataxin is the activation, in a way that is not yet understood, of sphingolipids. Sphingolipids, in turn, activate signal transduction pathways, like, in the example of photoreceptors, of Phosphoinositide-dependent kinase 1 and Myocyte enhancer factor 2 (Chen et al., 2016a,b). Moreover, murine mutants in Sod2 show increased lipid accumulation in their livers (Li et al., 1995; Chouchani et al., 2017), which we attribute to the inactivation of aconitase (**Figure 9**). It remains to be shown whether during normal physiology our hypothesis of two alternating mitochondrial states, one driving aerobic respiration, the other driving lipogenesis and heme biosynthesis, is valid or not.

#### PHYSIOLOGICAL RELEVANCE OF Fe-S AND MOCO ENZYMES IN DIFFERENT TISSUES

In this section we first look at the role of Fe-S enzymes during the life cycle of D. melanogaster, then focus on the main tissues of the adult fly where functions of Fe-S and Moco enzymes are known.

### Development, Growth, and Aging: Stem Cells and Cellular Differentiation

The life cycle of Drosophila is separated in distinct stages. Egg-laying follows the insemination of oocytes in the female, embryogenesis gives rise to the larva, which feeds and grows until entry into metamorphosis, at the end of which adult flies emerge (Demerec, 1950).  The decline in mitochondrial functions observed in late life, along with experiments showing that genetic manipulations leading to improved mitochondrial functions extended lifespan, suggested that mitochondrial metabolism governs the adult life span (Villee, 1948; Miquel, 1998; Fukagawa, 1999; Ross, 2000; Muller et al., 2007), but the finding that mitochondria isolated from old flies incubated with cytosol from young individuals restore respiration, whereas mitochondria from young individuals incubated with cytosol of old individuals fail to produce ATP questions this view (Vann and Webster, 1977; see also Sanz, 2016). On the other hand, there can be no doubt that defects in Fe-S cluster biosynthesis inhibit growth and dramatically shorten lifespan (Missirlis, 2003). It is also good to remember that the two sexes need to be considered separately when studying mitochondrial metabolism (Camus et al., 2012, 2015; Pomatto et al., 2017).

Increased glycolysis uncoupled from aerobic respiration is a characteristic of proliferative cells, for example during the early stages of Drosophila embryogenesis (Tennessen et al., 2011, 2014). This major metabolic switch is mediated through cell signaling (Thörig et al., 1981a,b; Markopoulou and Artavanis-Tsakonas, 1989; Homem et al., 2014; Barry and Thummel, 2016; Sieber et al., 2016; Slaninova et al., 2016; Mattila and Hietakangas, 2017). We already discussed that exit from the growth stage requires the concerted activity of Fe-S and heme enzymes in the prothoracic gland for ecdysone synthesis (Llorens et al., 2015; Palandri et al., 2015). Further, ecdysone is one of the signals inducing oxidative phosphorylation through the mitochondrial respiratory chain, sensed by neuroblasts and leading to their terminal differentiation (Homem et al., 2014) (see also Sen et al., 2013). Iron itself can directly influence stem cell differentiation, exemplified by hemocyte production in the lymph gland (Yoon et al., 2017). Thus, primary cell metabolism can define the fate of stem cells in a developing organism.

## The Germ Line: Is a Lineage of Quiescent Mitochondria Set Aside for Reproduction?

Stem cell differentiation in the ovary was found to depend on the mitochondrial ATP synthase (Teixeira et al., 2015). Surprisingly, in the female germarium, oxidative phosphorylation played no role in the early differentiation steps of the female germ line (Teixeira et al., 2015). Why are differentiating stem cells in the ovary and the larval brain different? One idea, first proposed by Allen (1996), is that the female germ line is defined as a carrier of quiescent (non-respiring) mitochondria (Cox and Spradling, 2003; de Paula et al., 2013a,b; Sieber et al., 2016). A key experiment to test this hypothesis is whether quiescent mitochondria can be detected throughout the life cycle in the female germ line (Allen and de Paula, 2013). We showed that quiescent mitochondria are observed in the female germ line within gonads of third instar larvae (**Figure 10**). However,

similar experiments have proven harder to perform in embryos due to impermeability of the Mitotracker Red and reduced spaghetti squash-EYFP fluorescence (LaJeunesse et al., 2004), although the laboratory of Richa Rikhy recently succeeded to image mitochondria in living embryos using new constructs (Chowdhary et al., 2017). Of note, these quiescent mitochondria would constitute a third mitochondrial state, not described in **Figure 9**, since they lack the oxidative phosphorylation complexes and hence a source of superoxide to protect the mitochondrial DNA. This line of research was also unfortunately disrupted at Queen Mary University of London (Allen and Missirlis, 2012; Ashworth, 2012; Horton, 2012); for further insightssee (Burrows, 2012; Mahul-Mellier et al., 2015; Lawrence, 2016; Edwards and Roy, 2017; Tsimilli-Michael and Haldimann, 2017).

Alan Spradling and co-workers have also described quiescent mitochondria in the oocyte (Cox and Spradling, 2003), but they have shown in addition an active mitochondrial membrane potential during stages 4–8 of oogenesis (Sieber et al., 2016). Insulin signaling acting through glycogen synthase kinase 3 is required to set mitochondria into a quiescent phase from stage 10 of oogenesis (Sieber et al., 2016). Is the finding of an active membrane potential during oogenesis sufficient to refute the theory of quiescent mitochondria? The first issue to resolve is whether mitochondria in the germarium respire or are quiescent. Our interpretation of the limited published data (Sieber et al., 2016) is that mitochondria in female germ cells are quiescent and this would offer an explanation for the unexpected finding that the early steps of the female germ line differentiation is independent of oxidative phosphorylation (Teixeira et al., 2015). However, another well-known phenomenon in the transmission of mitochondria through successive generations is bottleneck selection of functional mitochondria, which occurs during Drosophila oogenesis (Cox and Spradling, 2003, 2006; Hill et al., 2014; Ma et al., 2014; Chen Z. et al., 2015). It is therefore possible that the discovery by Ruth Lehmann and co-workers of ATP synthase induction in the first steps of such a differentiation program proceeds in order for mitochondria to test their ability to sustain a membrane potential during stages 4–8 of oogenesis as a selection of functional mitochondria that can be safely transmitted to the oocyte.

If Allen's hypothesis is proven correct, he may have resolved Darwin's still-standing question of why it is that two separate sexes evolved (Venton, 2013). The two sexes were required to keep a quiescent form of mitochondria in the resting gamete, while dispensing the mitochondria used by the motile gamete (DeLuca and O'Farrell, 2012; Politi et al., 2014). The mitochondrial DNA polymerase (an Fe-S enzyme, Stiban et al., 2014) participates in the elimination of paternal mitochondrial genomes in D. melanogaster by a mechanism that is not yet understood (Yu et al., 2017).

# Eye Pigment Formation

The study of heredity defines the birth of the field of Drosophila research, famously through observations of the color of their eyes (Morgan, 1910, 1911). The study of how eye pigments are formed, inspired by the drive to understand the genetic control of development (Beadle and Ephrussi, 1936; Lewis, 1978), gave rise to biochemical genetics. Progress in biochemistry (i.e., Hadorn and Mitchell, 1951) made clear early on that the Moco is required for pigment biosynthesis (Glassman and Mitchell, 1959; Hubby and Forrest, 1960). Progress in electron microscopy revealed impressive changes in cell biology occurring in eye color mutants (Nolte, 1961; Shoup, 1966; Sullivan and Sullivan, 1975) and, as discussed above, how Xdh reaches the pigment cells of the eye remains unsolved (Reaume et al., 1989, 1991).

#### Malpighian Tubules and Urate Excretion

Malpighian tubules are the major excretory organ in flies (Beyenbach et al., 2010). The primary role of Xdh is in purine catabolism, mostly taking place in the Malpighian tubules (Dickinson and Gaughan, 1981; Reaume et al., 1989). Zinc ions are implicated in mineral excretion through this organ (Chi et al., 2015; Yin et al., 2017), while the same genes that govern pigment granule formation in the eye are also required for the formation of zinc storage granules in the Malpighian tubules (Tejeda-Guzman et al., 2018). Finally, the Malpighian tubules also show strong Aox activity (Dickinson and Gaughan, 1981), although the physiological function of this activity remains to be shown (Marelja et al., 2014).

#### Muscles for Flight and Heartbeat