Fig 1.
G. intestinalis encodes components of the late ISC pathway.
(A) Schematic representation of mitosomal ISC pathway against the current model of the pathway as it occurs in aerobic mitochondria of fungi and animals. The early ISC pathway starts with a complex containing cysteine desulfurase IscS and its activator Isd11 with acylated ACP1. The complex is bound by IscU, on which the [2Fe-2S] cluster is built. Sulfur is released from cysteine by IscS and its transfer to the cluster is facilitated by frataxin, and the electrons for cluster formation are provided by reduced ferredoxin. Upon the formation of [2Fe-2S] cluster on IscU, a chaperone complex consisting of Hsp70 and HscB transfers the cluster to glutaredoxin 5 (Grx5). From Grx5, [2Fe-2S] cluster is either (I.) transferred to the target mitochondrial [2Fe-2S] apoproteins, (II.) exported to the cytosol as an enigmatic X-S compound or (III.) enters the late ISC machinery. The late ISC machinery starts with the transfer of two [2Fe-2S] clusters from Grx5 to a complex of IscA1, IscA2, and Iba57 where the [4Fe-4S] cluster is formed. The newly formed [4Fe-4S] clusters are delivered to apoproteins with the help of Nfu1 and Ind1. The precise role of BolA proteins remains unknown, but BolA1 was shown to interact with Grx5, while BolA3 interacts with Nfu1. The mitochondrial components that are missing in Giardia mitosomes are shown in gray. The early and late ISC pathways are distinguished by the background color. (B) Domain structure of GiGrx5, GiIscA2, GiNfu1, and GiBolA1. The respective sequence motifs and Pfam accession numbers are shown. (C) Protein sequence alignment of the identified GiBolA1 with the homologues from, Saccharomyces cerevisiae (Q3E793), Homo sapiens (Q9Y3E2), Plasmodium falciparum (Q8I3V0), Naegleria gruberi (D2V472) and Trypanosoma brucei (Q57YM0). BolA signature V/I/LHAL/I motif is highlighted. (D) Structure of GiBolA1 as predicted by AlphaFold2 [75], predicted structure of human BolA1 (HsBolA1) [27] is shown for comparison. (E) Maximum likelihood phylogenetic tree of 70 eukaryotic BolA1 paralogues shows that GiBolA1 and metamonad BolA homologues emerge from within a clade of mitochondrial BolA1 proteins. Summary of bipartition support values (1000 ultrafast bootstraps) greater than 80 or 95 are shown in open and closed circles, respectively.
Fig 2.
GiBolA1 is a mitosomal protein that specifically interacts with GiGrx5 and other ISC components.
(A) BAP-tagged GiBolA1, GiGrx5, GiNfu1 and GiIscA2 were expressed in G. intestinalis and the proteins were detected by anti-BAP antibody (green). The co-colocalization with mitosomal marker GL50803_9296 (magenta) is shown. The DIC image of the cell is shown in the inlet, the scale bar represents 5 μm. (B) Detection of BAP-tagged GiBolA1, GiGrx5, GiNfu1 and GiIscA2 in cellular fractions, lys–cell lysate, cyt—cytosol, HSP–high speed pellet fraction. (C) Protease protection assay of late ISC components and the markers of the outer mitosomal membrane (GiTom40) and the mitosomal matrix (GiIscU). High-speed pellets isolated from G. intestinalis expressing BAP-tagged GiBolA1, GiGrx5 GiIscA2 and GiNfu1 were incubated with 20 μg/ml trypsin and 0.1% Triton X-100. The samples were immunolabeled with antibodies against the BAP tag, GiTom40 and GiIscU. (D) Serial dilutions of Y2H assay testing the protein interactions between GiBolA1 and GiGrx5. The introduction of specific mutations of conserved residues (H90A GiBolA1 and C128A GiGrx5) abolished the interaction, double and triple dropout medium was used to test the presence of the plasmids and the interaction of the encoded proteins, respectively. (E) Affinity purification of the in vivo biotinylated GiBolA1 with the DSP-crosslinked interacting partners. (top right) Scheme of the in vivo biotinylation of the C-terminal BAP-tag of GiBolA1 by cytosolic BirA. (left) Volcano plot of the statistically significant hits obtained from the protein purification on streptavidin coupled Dynabeads. Components involved in ISC pathway are shown in bold letters.
Fig 3.
Characterization of BolA1 knockout (ΔbolA1) cell line.
(A) The ΔbolA1 cell line was tested for the presence of bolA1 gene and the integration of homologous recombination cassette (HRC) by PCR on gDNA, (B) the expression of bolA1 gene in ΔbolA1 cell line was tested by PCR on the cDNA, β-giardin was used as a control gene. (C) The number of mitosomes per cells in Cas9-expressing (n = 64) and ΔbolA1 cells (n = 107), the error bars of the box plot depict min to max values. (D) The list of predicted 40 Fe-S proteins in G. intestinalis includes only one mitosomal protein, [2Fe-2S] ferredoxin, that itself participates in the ISC pathway. All putative clients that require [4Fe-4S] clusters are localized in the cytosol or in the nucleus (S2 Table). (E) In vitro Fe-S cluster assembly assay in the mitosome-enriched high-speed pellet (HSP) fraction. The organelles or the cytosolic fraction were incubated in the reaction buffer supplemented with apoferredoxin (apo FDX), 35S-labeled L-cystein and ferrous ascorbate at 25°C for 60 min. The incorporation of 35S and the assembly of holoferreoxin (holo FDX) was analyzed on nondenaruring protein gel. The analysis of HSP from ΔbolA1 cell line showed that the activity was not abolished in the absence BolA1. (F) Incorporation of 55Fe to G. intestinalis proteins after 72 h incubation with radioactive iron isotope in the form of ferric citrate. Comparisons of control and ΔbolA1 cell extracts show comparable levels of iron incorporation.
Fig 4.
Proteomic analysis of late ISC pathway.
BAP-tagged GiGrx5, GiNfu1 and GiIscA2 were in vivo biotinylated by cytosolic BirA and purified on streptavidin-coupled Dynabeads upon crosslinking by DSP. (A-C) Volcano plots depict the significantly enriched proteins that co-purified with (A) GiNfu1, (B) GiGrx5 and (C) GiIscA2. (D) Heatmap of combined significantly enriched proteins for all four late ISC components, (E) Digitonin solubilization of the mitosomes shows differential release of GiIscA2 over GiIscU, P -pellet fraction (retained protein), S–supernatant (released protein). Exemplary western blot of four independent experiments is shown, the error bars show standard deviation.
Fig 5.
Adaptation of the ISC pathway in anaerobic mitochondria of Metamonada.
The presence/absence of the ISC components in Metamonada supergroup Absence from the transcriptome means that only transcriptomic data were available for the analysis.