Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis

Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron–sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe–2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron–sulfur protein assembly (CIA) pathway includes the essential Cfd1–Nbp35 scaffold complex that assembles a [4Fe–4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes.

Monothiol glutaredoxins bind a [2Fe-2S] cluster in a bridging fashion. The iron atoms are coordinated by the cysteine in the active site CGFS (in yellow) of the protein and a cysteine from bound glutathione. Monothiol glutaredoxins from bacteria and Grx3 and Grx4 from yeast form homodimers and it has been proposed that switching from dimeric to monomeric conformation releases the [2Fe-2S] cluster to acceptor proteins. In human, the holo-GLRX5 is tetrameric, whereas the metal free protein is also monomeric [10][11][12][13] . The amino acids involved in inter-subunit interaction in human Grx5 are shown in green. The amino acids in pink stabilize a loop in the tetrameric structure that shields the [2Fe-2S] cluster. In light blue and bold: essential residues for the biological activity of yeast Grx5 14 . The glutaredoxin domain (PF00462) is underlined in blue. The N-terminal thioredoxin-like domain (SSF52833) identified in E. cuniculi and E. intestinalis glutaredoxins using HHPred (http://toolkit.tuebingen.mpg.de/hhpred) 15 is highlighted in red. A CDD search 16 identified a thioredoxin-like domain (cd02984) in the protein from C. parvum (red). By contrast, no thioredoxinlike domain was identified in the corresponding segment of the T. hominis and G. intestinalis sequences. IYPTKFLVYQVIYSDYLERLSCRAFYTVLSRRWEITLQEEGLNSRFKNL-
The ABC signature motif (LSGG), also called the C-loop, which is present in the nucleotide-binding fold, is labelled in grey 22 . The D-loop (SALD), which interacts with the Walker A motif is labelled in teal 23 . The conserved histidine that is proposed to be involved in the catalytic reaction is labelled in light grey 24 . The residues identified in Saccharomyces cerevisiae that interact with glutathione are labelled in yellow. The star (★) corresponds to the human Atm1 E433K mutation found in patients with X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A) 25 .

e. P-loop NTPase Cfd1 and Nbp35
Cfd1_Tra_hom - SQ GVEGETIHVSST - - LGALVVTTPQAVSVGDVRRELTFCRKTGLRVMGIVENMSGFTCPHCTECT The major structural difference between Cfd1 and Nbp35 is the presence in Nbp35 of an N-terminal Cys-X13-Cys-X2-Cys-X5-Cys ferredoxin-like motif (labelled in red) coordinating a [4Fe-4S] cluster [26][27][28] . The insertion of this cluster depends on electron transfer from the Tah18-Dre2 complex 29 . The genome of E. histolytica lacks homologues of Tah18 and Dre2, which is consistent with the loss of the ferredoxinlike motif in E. histolytica Nbp35 30 . Cysteine residues in the CX18CPXCX2C (Cfd1) and CX18CX2CX38C (Nbp35) motifs that are conserved at the C termini of yeast Cfd1 and Nbp35 homologues are labeled in yellow and dark green respectively. The two central cysteine residues of both motifs, which are conserved in microsporidian sequences, are essential for yeast viability and for maturation of cytosolic and nuclear Fe/S proteins. These motifs also have a critical role in Cfd1-Nbp35 complex formation 26,28,31 . Cfd1 and Nbp35 are classified as Mrp-like proteins that belong to the Mrp/Nbp35 subfamily of P loop NTPases. Nucleotide binding and/or hydrolysis is apparently critical for loading an Fe/S cluster onto the Cfd1-Nbp35 complex. The consensus Walker A motif of the Mrp family and the ENMS motif are labeled in pink and light blue respectively. The Asn in the ENMS motif is predicted to form a contact with the adenine from ATP 32 . The Walker B motif with a conserved Gly residue (typically, within the signature hhhhDxxG, where h is a hydrophobic residue) is labeled in green.

f. WD40 protein Cia1
Cia1_Tra_hom Cia1 belongs to the WD40-repeat protein family. WD40 repeats are conserved domains of approximately 44-60 residues that typically contain the GH dipeptide 11-24 residues from its N terminus and the WD dipeptide at the C-terminus. The WD repeat combines a conserved core structure with variable regions that are probably surface-exposed. Most WD proteins contain a cluster of at least 7 or more copies of WD-repeats with as many as 16 but as few as four. A WD protein forms a propeller-like structure with several blades where each blade is composed of a four-stranded anti-parallel β-sheet. Each WD40 sequence repeat forms the first three strands of one blade and the last strand in the next blade. The proposed common function of this protein family is to coordinate the assembly of multi-protein complexes by functioning as a docking site for other proteins. Residues on the top and bottom surface of the propeller are proposed to coordinate interactions with other proteins and/or small ligands 33,34 . The 3D structure of the Cia1 protein from S. cerevisiae shows the typical architecture of other WD40-repeat proteins 35,36 . The β propeller structure contains three potential interacting surfaces: The top, the bottom and the circumference 35,36 . The WD40 repeats are shadowed in yellow. The seven WD40 repeats in S. cerevisae were obtained from Unipropt KB: http://www.uniprot.org/uniprot/Q05583 and the WD40 repeats from the other organisms were obtained from SMART: http://smart.embl-heidelberg.de/. Note that Tra_hom has four inferred WD repeats, Enc_cun and Enc_int have five WD repeats, Gia_int and Ent_his have six WD repeats. The arginine (R) in green is critical for the function of Cia1 in yeast 36 and is conserved in all of the microsporidian sequences. In bold are the invariant residues present also in the microsporidian sequences.

h. Hydrogenase-like Nar1
Nar1_Tra_hom LYGRLS TAATADPYHSDYIEVNACPGACMNGGGLLNGEQNSLKR QKEWQKEVDEAYFSG-----DESGSRAQDESLDLVVDGISPSHIRNVLTH Nar1_Hom_sap GMVRAEAPEDAPGVQ  Nar1 proteins are related to Fe-only hydrogenases 40 and contain two-conserved cysteine motifs. One is located at the N-terminus and the second is distributed between the central part of the protein and its Cterminus, the cysteine residues forming these motifs are coloured in red and blue, respectively. In yeast, each cysteine motif coordinates a [4Fe-4S] cluster and both are essential for the assembly of cytosolic Fe/S proteins 41 . It has been shown that the C-terminal Fe/S cluster is stably bound to the protein and its assembly depends on the Fe/S cluster from the N-terminal cysteine motif 42 . A conserved C-terminal tryptophan characteristic of the Nar1 protein family is highlighted in yellow. -  29 . In human and yeast the C-terminus of the proteins are connected to an N-terminal S-adenosylmethionine (SAM) methyltransferase-like domain which is not known to bind SAM 45 . The SAM-like domain is not present in the microsporidian Dre2-like proteins and was not detected using CDD at the NCBI 16 . Notably, residues 1-172 of this region are known to be important for Dre2 function in Fe/S protein biogenesis, yet the region is not essential for yeast cell viability [43][44][45] .

i. Fe/S protein Dre2
In yellow: Acidic and serine (E, D and S)-rich patch identified in human and yeast Dre2 43 . The E, D and S residues in this region of microsporidian proteins were also labelled in yellow for comparison. Tah18_Neu_cra  Components of the mitosomal ISC pathway have originated from the mitochondrial endosymbiont. The CIA pathway appears largely bacterial in character, and not archaeal as might be expected given that the host for the mitochondrial endosymbiosis is now thought to have descended from an Archaeon. By contrast, important nuclear and cytosolic Fe/S proteins do appear to have originated from an Archaeon. Monophyly of eukaryotic sequences, including those from microsporidians, is generally observed, suggesting that there is strong negative selection against gene replacement and reflecting the important roles that Fe/S proteins play in eukaryotic physiology.  Note that the region that can be aligned between eukaryotes and prokaryotes is quite short for this protein (122 amino acids), and the specific relationships between the eukaryotes and any particular prokaryotic group are therefore tentative. Although this tree is poorly resolved, the best BLAST hits of eukaryotic Dna2 are to sequences from the Haloarchaea; this is perhaps consistent with an archaeal origin for this gene, given the presence of good homologues in these and other Euryarchaeota. The Giardia and Plasmodium sequences included here are the most likely Dna2 candidates in these species, although the tree topology weakly excludes them from the otherwise monophyletic eukaryotic clade.  targeting signal, but this has also been observed for T. hominis mitosomal Hsp70 which is nevertheless targeted to the organelle. ThAtm1_1 and ThAtm1_2 also contain most of the recognized glutathionecoordinating residues 25 . Other conserved motifs within the NBD include the Walker A motif and the preceding conserved basic amino acid 20 ; the Q-and C-(signature) loops which are part of the ATPbinding motif 22 ; the Walker B motif followed by a glutamate residue which acts as a catalytic base 18 ; the D-loop which interacts with the Walker A motif 23 , and a conserved histidine that is proposed to be involved in the catalytic reaction 24 . The TMDs were predicted with the TOPCONS server b, Immunofluorescence microscopy of paraformaldehyde-fixed (2.5% PFA in PBS at room temperature for 10 min followed by permeabilization in 0.2% Triton X-100 in PBS for 10 min, top row) or methanol:acetone-fixed (1:1 at -20°C for 10 min, bottom row) RK cells infected with T. hominis (Th) using a rat antibody to T. hominis mitosomal Hsp70 (ThHsp70, green), and a rabbit antibody to the T.
Merged images are shown on the right. Scale bars correspond to 2 µm.