Chlamydomonas ATX1 is essential for Cu distribution to multiple cupro‐enzymes and maintenance of biomass in conditions demanding cupro‐enzyme‐dependent metabolic pathways

Abstract Copper (Cu) chaperones, of which yeast ATX1 is a prototype, are small proteins with a Cu(I) binding MxCxxC motif and are responsible for directing intracellular Cu toward specific client protein targets that use Cu as a cofactor. The Chlamydomonas reinhardtii ATX1 (CrATX1) was identified by its high sequence similarity with yeast ATX1. Like the yeast homologue, CrATX1 accumulates in iron‐deficient cells (but is not impacted by other metal‐deficiencies). N‐ and C‐terminally YFP‐ATX1 fusion proteins are distributed in the cytoplasm. Reverse genetic analysis using artificial microRNA (amiRNA) to generate lines with reduced CrATX1 abundance and CRISPR/Cpf1 to generate atx1 knockout lines validated a function for ATX1 in iron‐poor cells, again reminiscent of yeast ATX1, most likely because of an impact on metalation of the multicopper oxidase FOX1, which is an important component in high‐affinity iron uptake. We further identify other candidate ATX1 targets owing to reduced growth of atx1 mutant lines on guanine as a sole nitrogen source, which we attribute to loss of function of UOX1, encoding a urate oxidase, a cupro‐enzyme involved in guanine assimilation. An impact of ATX1 on Cu distribution in atx1 mutants is strikingly evident by a reduced amount of intracellular Cu in all conditions probed in this work.


| INTRODUCTION
Copper (Cu) is an important trace element in all kingdoms of life, because it serves as an essential cofactor in enzymes that participate in diverse metabolic pathways, from photosynthesis and respiration to oxidative stress protection and iron acquisition. Although a particular quota of Cu is required for fully functioning metabolism, excess intracellular Cu ions may result in damage of macromolecules through redox or oxygen chemistry with negative consequences for the cell. Cu assimilation and sequestration is therefore tightly regulated by orchestrated function of Cu transporters, ligands, and chaperones.
A general pathway for Cu metabolism has been developed based on discoveries in several systems. Cu enters the cell through a high affinity Cu(I) uptake system that includes CTR/COPT family proteins (Puig & Thiele, 2002). After entry, cytosolic chaperone proteins (of which ATX1 is a prototype) are responsible for subsequent Cu transfer to key metabolic Cu proteins Rosenzweig & O'Halloran, 2000;Shi et al., 2021). More recently, GSH is implicated as a Cu ion carrier between the importers and metallochaperones (Miras et al., 2008).
In Saccharomyces cerevisiae, where Cu metabolism is well dissected, three Cu chaperones compete for Cu entering the cell, delivering them to their specific targets (Valentine & Gralla, 1997). yATX1 is a small protein (73 aa) that delivers Cu to the trans-Golgi network for incorporation into membrane-bound and secreted Cu proteins (Lin & Culotta, 1995). yATX1 was discovered originally as a suppressor of oxygen toxicity damage in yeast strains lacking SOD1 function, hence its name Atx for antioxidant (Lin & Culotta, 1995). The Cu binding motif, MXCXXC, is present also in many ATP-dependent Cu exporters (Cu P-type ATPases). yATX1 transfers Cu from the inner plasma membrane to the secretory pathway where Cu P-type ATPases such as Ccc2 are localized for sequestering Cu into the secretory pathway (Hung et al., 1997;Lin et al., 1997). Ccc2 directly interacts with ATX1, enabling Cu transfer to Ccc2 for translocating Cu coupled to ATP hydrolysis (Arnesano et al., 2002;Pufahl et al., 1997). Notably, all Cu-dependent enzymes from the secretory pathway are loaded with Cu ions in the Golgi. One such enzyme is the cupro-protein FET3, a plasma membrane spanning multicopper ferroxidase, which is required for high-affinity iron uptake in yeast . Accordingly, yeast atx1 mutants are unable to load Cu into the active site of FET3, resulting in defective iron uptake and iron deficiency . Yeast ctr1 mutants defective in Cu import and yeast ccc2 mutants defective in transferring Cu to the secretory pathway also display an iron deficiency phenotype that can be rescued by excess Cu (Askwith & Kaplan, 1998;Dancis et al., 1994).
Atx-like Cu chaperones are found with high sequence similarity in most eukaryotes, including mammals, algae, and land plants. The human ATX1 homologue (HAH1) was shown to rescue the growth defect of yeast atx1 mutants in iron-poor medium by restoring Cu incorporation into newly synthesized FET3 . HAH1 directly transfers Cu ions to the Cu P-type ATPases ATP7A and ATP7B, which are involved in Cu delivery to the secretory pathway (Banci et al., 2005). Two ATX1 homologues named Cu CHaperone (CCH) and AtATX1 have been analyzed from Arabidopsis. Both proteins, CCH as well as AtATX1, complement the iron conditional growth defect of yeast axt1 mutants (Himelblau et al., 1998;Puig et al., 2007). Direct interaction with the plant Cu P-type ATPase RAN1 was established for AtATX1 (Puig et al., 2007). Mutants defective in AtATX1 are hypersensitive to both Cu excess and Cu deficiency conditions (Shin et al., 2012), indicating the importance of this protein for Cu homeostasis.
Like other algae, the eukaryotic green alga Chlamydomonas reinhardtii, referred to as Chlamydomonas hereafter, has a reduced Cu quota, perhaps because of the lack of a Cu/ZnSOD (Asada et al., 1977;Omelchenko et al., 2010). The genome of Chlamydomonas encodes a single ATX1 homologue, CrATX1 ( Figure 1). Transcriptional profiling indicates that CrATX1 expression is increased in response to iron deficiency in Chlamydomonas along with other components of Fe assimilation like FOX1, FTR1, FER1, and FEA1 (La Fontaine et al., 2002). An orthologous function to yeast ATX1 and human HAH1 was suggested based on functional complementation of the yeast ATX1 strain and the relationship of multicopper oxidase CrFOX1 to yFET3 (La Fontaine et al., 2002;Urzica et al., 2012).
In this work, we extend the original study to document selective ATX1 expression and protein accumulation in response to iron and nitrogen nutrition (but not Cu or Zn), localize YFP-tagged ATX1 to the cytoplasm, and assess function in Chlamydomonas by reverse genetics under conditions where cupro-proteins are required: Fe limitation and guanine as a sole N-source, requiring, respectively, cupro-enzymes FOX1 and UOX1 encoding a multicopper Fe(II) oxidase and urate oxidase. We conclude that CrATX1 is the cytosolic Cu chaperone responsible for Cu delivery from CTR-like Cu importers and/or GSH at the plasma membrane to the secretory pathway. But surprisingly, our work indicates that ATX1 might have an additional, more general role in copper homeostasis, as atx1 mutants have a significantly reduced copper content in all conditions that were interrogated in this work.
F I G U R E 1 ATX1 is a conserved and widespread protein. The evolutionary history was inferred by using the maximum likelihood method and JTT matrix-based model (Jones et al., 1992). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). When the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) exceeded 50%, they are shown next to the branches. This analysis involved 22 amino acid sequences. There were a total of 90 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018) 2 | EXPERIMENTAL 2.1 | Strains and culture conditions An miRNA targeting Chlamydomonas ATX1 was designed according to (Molnár et al., 2009;Schmollinger et al., 2010)  instead of Hutner's trace elements according to Kropat et al. (2011).
For all experiments with atx1 amiRNA strains, a modified TAP medium (TAP (NO 3 )) was used, where nitrate was substituted instead of ammonium as the sole nitrogen source to induce the artificial microRNA.

| Generation of atx1 KO strains using CRISPR/ LbCpf1
CC-425, a cell wall reduced arginine auxotrophic strain, was used for transformation with an RNP complex consisting of a gRNA targeting a PAM sequence in intron1 of ATX1 and LbCpf1 as described in Ferenczi et al. (2017) and shown in Figure 5 with the following modifications: Cells were grown to a density of 2 Â 10 6 cells per milliliter and counted using a Coulter counter. Cells (2 Â 10 7 ) were collected by centrifugation (5 min, 1500 g) and resuspended twice in Max Efficiency Transformation Reagent (1 ml), followed by resuspension in

| Antibody production and protein analyses
Antibodies targeting ATX1 were produced by Covance, Inc. by immunization using the subcutaneous implant procedure for rabbits on a 118-day protocol with synthetic peptide Ac-

| ATX1 is conserved in most algae
Because the inventory of cupro-proteins in algae is distinct from that in other eukaryotes (yeast, human, land plants), we sought to understand the role of the Chlamydomonas orthologue of the ATX1 copper chaperone. We recovered ATX1 sequences from various eukaryotic organisms using yATX1 as a query (Figure 1) (Himelblau et al., 1998;Puig et al., 2007). All algal ATX1 proteins share the MXCXXC domain that is required for Cu binding and a defining characteristic of the ATX1 family of Cu chaperones (Supplemental Figure 1). F I G U R E 4 Subcellular localization of YFP, an N-terminal YFP-ATX1 fusion protein as well as a C-terminal ATX1-YFP fusion protein. Confocal microscopy of UVM11 control (untransformed), ATX1 protein fused with YFP at the N-or Cterminus (magenta), and YFP expressed without a fusion protein driven by the constitutive PSAD promoter, respectively. Chlorophyll autofluorescence is shown in green (Chl) and bright-field images (DIC) are shown to depict the cell and the chloroplast, respectively. All strains were grown in medium supplemented with 1 μM Fe. Scale bars correspond to 5 μm. At least 10 individual cells derived from two independent cultures were imaged and are shown in Supplemental  Figures 3-6 are therefore not strictly co-expressed; simultaneous expression is limited to conditions involving reduction of Fe in the growth medium.

| ATX1 is predominantly expressed in conditions in which iron is scarce
To test the impact of the change in ATX1 mRNA abundance, we analyzed the abundance of the corresponding polypeptide as a function of iron nutrition. Chlamydomonas cells were grown in TAP medium supplemented with 20, 1, and 0.2 μM iron, corresponding to replete, deficient, and limited growth, respectively (Glaesener et al., 2013). We recapitulated previously reported phenotypic differences in growth, including chlorosis and growth arrest on media containing 1 and 0.2 μM Fe as compared with 20 μM Fe (Figure 2b).
Chlorosis was particularly strong in iron-limited cultures, likely due to complete exhaustion of the iron source from the growth medium . Immunoblot analysis indicated that ATX1 is iron-conditionally expressed (Figure 2b), which is consistent with a role for Chlamydomonas ATX1 in Cu delivery to the secretory pathway for FOX1 biosynthesis.

| ATX1 is a cytosolic protein
To distinguish ATX1 localization, we tagged the protein with a fluorescent reporter (YFP) at the N-terminus and at the C-terminus and expressed the respective fusion proteins from a strong, constitutive promoter (PSAD; Figure 3a) into strain UVM11, which expresses nuclear transgenes efficiently (Neupert et al., 2009). We identified a line for each of the constructs that showed a signal against both F I G U R E 5 Strains with reduced ATX1 abundance using artificial microRNA constructs and atx1 KO lines using CRISPR/Cpf1-mediated gene editing. (a) Physical map of the ATX1 gene and target site for the amiRNA as well as the PAM sequence for Cpf1 recruitment. (b) CRISPR/Cpf1and ssODN-mediated gene editing resulted in the insertion of two in-frame stop codons into ATX1.
(c) amiRNA lines 1, 2, atx1-1, and atx1-2 grown in TAP supplemented with 0.2 μm Fe (limited) showed reduced ATX1 mRNA abundance in comparison with the respective Fe-limited reference strains (ATX1). (d) Soluble protein from Fe-limited cultures was separated by 15% SDS-PAGE, transferred to nitrocellulose membranes, and immunodetection was performed using antisera against ATX1 to confirm reduced ATX1 protein abundance in atx1 amiRNA lines and absence of ATX1 protein in atx1 mutants. Coomassie blue (CB) stain was used as loading control ATX1 and GFP antiserum with a band in each case at the expected size of the fusion protein (Figure 3b). The analysis using the GFP antiserum showed no other visible bands below the size of the fusion protein, indicating that there were no cleavage products and no expression of YFP without ATX1 in the transformants, which would potentially interfere with localization analysis using confocal microscopy ( Figure 3b). We chose a line expressing YFP alone, the abovementioned line expressing an N-terminal YFP-ATX1 fusion protein, as well as a line expressing a C-terminal ATX1-YFP fusion protein ( Figure 3) for further analyses by direct live cell imaging. Notably, using chlorophyll autofluorescence to visualize the chloroplast, we conclude that both YFP-ATX1 fusion proteins are localized to the cytoplasm, consistent with ATX1's proposed role in Cu trafficking from the inner plasma membrane to the trans-Golgi network ( Figure 4).

| ATX1 is required for growth in low iron conditions
To validate the function of ATX1 in Chlamydomonas, we designed an artificial microRNA (amiRNA) targeting the 3 0 coding sequence of the CrATX1 gene (Figure 5a, red bar) (Molnár et al., 2009;Schmollinger et al., 2010). We initially screened candidate ATX1 knockdown lines based on reduction of ATX1 mRNA as compared with control lines containing an empty vector that lacks the target region against CrATX1 (ATX1). The candidate knockdown lines were grown in 0.2 μM Fe to maximally induce ATX1 expression to improve signal to noise in the screen. We chose two lines that showed reduced ATX1 mRNA abundance, which we named ami-atx1(1) and ami-atx1 (2) (Figure 5c). Compared with empty vector-transformed control strains, the abundance of ATX1 was reduced to about 50% and 25% in Fe-limited ami-atx1(1) and ami-atx1(2), respectively (Figure 5c). In parallel, we generated atx1 knockout lines using CRISPR/ CPF1-mediated gene editing adapted from previous protocols using homology-directed DNA replacement and the enzyme LbCpf1 in combination with a selection marker (Ferenczi et al., 2017;Greiner et al., 2017). ATX1 has a PAM (protospacer adjacent motif) target sequence for LbCPF1 recruitment within its intron ( Figure 5, green bar) that allowed gene editing at the downstream Cpf1 cleavage site within the second exon of ATX1. We used ssODNs as a repair template to introduce two in-frame stop codons within the second exon of the ATX1 gene (Figure 5b). Sequencing of the PCR product spanning the gene editing site confirmed the presence of the two stop codons in two atx1 lines, depicted atx1-1 and atx1-2 (Figure 5b).
If ATX1 functions in Cu trafficking toward the secretory pathway, atx1 mutant lines would be affected in FOX1 metalation and hence display a phenotype in iron-poor medium where FOX1 function is required (Terzulli & Kosman, 2009). To test this, we grew ami-atx1(2), atx1-1, and atx1-2 and corresponding control lines in iron-replete, iron-deficient, and iron-limiting media. Neither the amiRNA line nor atx1 mutants showed a growth defect in iron replete conditions, which is expected because ATX1 and its proposed client FOX1 are barely expressed in this situation ( Figure 6).
On the other hand, in iron-limiting conditions, both ami-atx1(2) and atx1 mutants showed reduced growth as compared with control lines ( Figure 6). When we measured the Fe content of each strain by ICP-MS/MS, we noted that all strains showed a reduction of the Fe content down to a basal (presumed essential) level in Fe-poor conditions. Because Fe is a growth-limiting nutrient, Fe-limited cells cease growth when the basal essential level of iron cannot be supported. Accordingly, atx1 mutant and ami-atx1 lines have reduced growth but maintain the minimal essential Fe quota comparable with wild-type lines (Figure 7a).
F I G U R E 6 Iron nutrition-dependent growth defect is exacerbated in ATX1, atx1-ami lines and atx1 mutants. ATX1 reference lines (black, filled symbols), atx1-ami(2) (white symbols), and atx1 mutants (orange-filled symbols) were grown in either iron-replete TAP medium (20), in Fedeficient (1), or in Fe-limited (0.2) growth medium as indicated. Cell counts were obtained at 24-h intervals. Shown are averages and standard deviation from three independent grown cultures. Below each panel are pictures of flasks taken 4-days post-inoculation. Asterisks indicate significant changes between atx1-ami lines and reference strains or between both atx1 mutants and respective reference strains at the same time point, respectively. Significant differences were determined by one-way ANOVA followed by Holm-Sidak, p-value α ≤ 0.05 We noticed before that Fe-deficient cells slightly upregulate components of the Cu assimilation pathway, likely due to a link between both pathways via FOX1 (Dancis et al., 1994). Accordingly, the copper content of cells grown on growth medium lacking iron is increased twofold compared with iron-replete grown cells (Figure 7b). On the other hand, Cu content was significantly lower in ami-atx1 lines as well as in atx1 mutants as compared with their respective reference lines in all conditions that were analyzed (Figure 7b). These data indicate a second phenotype in atx1 mutants, namely, a remarkable impact on Cu assimilation and accumulation.

| ATX1 is important for purine assimilation in nitrogen-poor conditions
Urate oxidase, an enzyme involved in purine metabolism (Figure 8), is also a Cu enzyme, making it another candidate client of ATX1 (Alamillo et al., 1991). In accordance with our expectations, we noted that UOX1, encoding urate oxidase, and ATX1 transcripts both increase transiently under N deficiency (Figure 8a). Notably, the observed increase in ATX1 transcript abundance upon N starvation is paralleled by an increase in the corresponding polypeptide (Figure 8b).
F I G U R E 7 Iron and copper content in atx1 mutants and atx1-amiRNA lines. (a) Iron and (b) copper content of empty vector (ATX1, black) and atx1-ami lines (atx1-ami(1) and atx1-ami (2) To test whether urate oxidase is an ATX1 client, we grew the reference wild-type strains and each atx1 mutant on guanine versus allantoin as a sole nitrogen source (Figure 8c). Both guanine and allantoin are taken up by Chlamydomonas, but guanine metabolism requires UOX1 whereas allantoin metabolism does not (Lisa et al., 1995;Merchán et al., 2001;Piedras et al., 1998). Strikingly, after 72 h of growth, atx1 mutants showed a visible growth defect on guanine but not allantoin as compared with the corresponding reference lines ( Figure 9), with significant less biomass accumulation as determined by chlorophyll content and cell counts (Figure 9b). We conclude that ATX1 is required for UOX1 function, most likely for its metalation.
In Chlamydomonas, multiple pathways of Cu trafficking emerge from the high-affinity, CTR-type Cu import system (Page et al., 2009).
Cu(I) taken up by CTRs is delivered to cytosolic Cu chaperones, perhaps via glutathione as an intermediate carrier (Miras et al., 2008), for redistribution to the mitochondria, to the chloroplast, and to the secretory pathway (Valentine & Gralla, 1997 First, both FOX1 and ATX1 are conditionally expressed as a function of iron nutrition. Cells grown in iron-limited media are characterized by chlorosis and growth arrest, and this phenotype is exacerbated in atx1 mutants. Yet, strains with reduced or even no ATX1 abundance have the same iron content as do wild-type cells. This phenotype mirrors that noted in fox1 knockdown lines (Chen et al., 2008). This is because Fe is a growth-limiting nutrient. When the minimal Fe quota is not met, no further growth can occur, and ami-atx1 lines as well as atx1 mutants recapitulate the iron conditional growth defect of fox1 knockdown lines. Because both fox1 and atx1 mutant strains do grow, albeit poorly, in iron-poor conditions, we propose that there must be a Cu-independent alternative system to facilitate iron uptake. The ZIP family transporters IRT1 and IRT2 are likely candidates Chen et al., 2008). This is further supported by the observation that Cu-deficient cells are also not secondarily iron deficient, despite FOX1 lacking its Cu cofactor in this situation (Kropat et al., 2015).
Second, we see a growth phenotype when cells are grown in media with guanine as the sole nitrogen source. In this situation, cells are dependent on purine assimilation via UOX1, which is also metalated in the secretory pathway (Alamillo et al., 1991). Again, the atx1 mutants show reduced growth as compared with reference wild-type lines. The third line of evidence is the reduced copper content of atx1 mutants, which speaks to its role in Cu(I) assimilation.
By expressing an N-terminal and a C-terminal YFP-ATX1 fusion protein in Chlamydomonas, we were able to demonstrate that CrATX1 is localized within the cytoplasm, consistent with the function of ATX1 in other organisms. Chlamydomonas encodes several Cudependent P-type ATPases , one of which, CTP1, is proposed to serve as the yeast Ccc2 orthologue, pumping Cu(I) into the lumen of the Golgi compartment. Like ATX1, CTP1 expression is also induced in Fe-starved cells (La Fontaine et al., 2002). This work documents that the loading of Chlamydomonas FOX1 and potentially also urate oxidase is dependent on this Cu(I) delivery pathway.