The iron-sulfur scaffold protein HCF101 unveils the complexity of organellar evolution in SAR, Haptista and Cryptista

Abstract

Background Nbp35-like proteins (Nbp35, Cfd1, HCF101, Ind1, and AbpC) are P-loop NTPases that serve as components of iron-sulfur cluster (FeS) assembly machineries. In eukaryotes, Ind1 is present in mitochondria, and its function is associated with the assembly of FeS clusters in subunits of respiratory Complex I, Nbp35 and Cfd1 are the components of the cytosolic FeS assembly (CIA) pathway, and HCF101 is involved in FeS assembly of photosystem I in plastids of plants (chHCF101). The AbpC protein operates in Bacteria and Archaea. To date, the cellular distribution of these proteins is considered to be highly conserved with only a few exceptions. Results We searched for the genes of all members of the Nbp35-like protein family and analyzed their targeting sequences. Nbp35 and Cfd1 were predicted to reside in the cytoplasm with some exceptions of Nbp35 localization to the mitochondria; Ind1was found in the mitochondria, and HCF101 was predicted to reside in plastids (chHCF101) of all photosynthetically active eukaryotes. Surprisingly, we found a second HCF101 paralog in all members of Cryptista, Haptista, and SAR that was predicted to predominantly target mitochondria (mHCF101), whereas Ind1 appeared to be absent in these organisms. We also identified a few exceptions, as apicomplexans possess mHCF101 predicted to localize in the cytosol and Nbp35 in the mitochondria. Our predictions were experimentally confirmed in selected representatives of Apicomplexa (Toxoplasma gondii), Stramenopila (Phaeodactylum tricornutum, Thalassiosira pseudonana), and Ciliophora (Tetrahymena thermophila) by tagging proteins with a transgenic reporter. Phylogenetic analysis suggested that chHCF101 and mHCF101 evolved from a common ancestral HCF101 independently of the Nbp35/Cfd1 and Ind1 proteins. Interestingly, phylogenetic analysis supports rather a lateral gene transfer of ancestral HCF101 from bacteria than its acquisition being associated with either α-proteobacterial or cyanobacterial endosymbionts. Conclusion Our searches for Nbp35-like proteins across eukaryotic lineages revealed that SAR, Haptista, and Cryptista possess mitochondrial HCF101. Because plastid localization of HCF101 was only known thus far, the discovery of its mitochondrial paralog explains confusion regarding the presence of HCF101 in organisms that possibly lost secondary plastids (e.g., ciliates, Cryptosporidium) or possess reduced nonphotosynthetic plastids (apicomplexans).

HCF101 is a novel component of the CIA cytosolic iron-sulfur synthesis pathway in the human pathogenToxoplasma gondii

“…Another clue of evolutionary specialization comes from the different localization of protein isoforms in different species, hinting for a possible association to different Fe-S cluster biogenesis machineries. While HCF101 was initially characterized as a chloroplast-associated protein with a specialized function in maintaining PSI homeostasis, a recent study suggested a wider diversity in localization for HCF101 paralogs across eukaryotes, especially in alveolates, with isoforms potentially localizing to the mitochondrion in Tetrahymena or in the cytosol in T. gondii [23]. Our study confirms that although T. gondii harbors a plastid, TgHCF101 is expressed in the cytosol (Fig.…”

“…We performed homology searches in the http://www.ToxoDB.org database to retrieve members of the Mrp/NPB35 family encoded by the T. gondii genome. Besides TgNBP35, that has an unusual mitochondrial localization in T. gondii [20,23], we identified TGGT1_318590 as a potential homologue of HCF101. A broader phylogenetic analysis that included members of the Mrp/NPB35 family from a wide variety of eukaryotes (Table S1), revealed that this putative T. gondii homologue clearly segregates with members of the HCF101 group (Fig.…”

“…The SUF pathway being essential for the survival of the parasite and absent from its mammalian host, its components and client proteins are particularly attractive as potential drug targets [21], which prompted us to look for Fe-S proteins absent from mammals but present in plant and apicomplexan parasites. Investigating the Fe-S cluster assembly components in protists, which are highly divergent phylogenetically from the canonical yeast or mammalian cell models, can bring interesting insights into the evolution of this molecular machinery [22,23].…”

TheToxoplasma gondiimitochondrial transporter ABCB7 is essential for cytosolic iron-sulfur cluster biogenesis and protein translation

“…For example, the cytosolic scaffold NBP35, which plays a central role in assembling cytosolic clusters, was found to be bound to the outer surface of the mitochondria (23), whereas in other model systems it is a soluble cytosolic protein, with one study suggesting a nuclear localisation (50). The exact reason for NBP35's re-localisation remains unclear, but it may highlight the connection between the CIA and ISC pathways in apicomplexan parasites and reflects the complex evolutionary history of these proteins in eukaryotes (51)(52)(53) Studies in a wide array of organisms clearly show that the role of the ABCB7 transporter in connecting the mitochondrial ISC and cytosolic CIA pathway is conserved (3). Downstream cytosolic Fe-S proteins are dependent on the cysteine desulfurase activity of the mitochondrial NFS1 enzymes, and the resulting sulfur intermediate that is transported out of the mitochondrial matrix by ABCB7.…”

“…For example, the cytosolic scaffold NBP35, which plays a central role in assembling cytosolic clusters, was found to be bound to the outer surface of the mitochondria (23), whereas in other model systems it is a soluble cytosolic protein, with one study suggesting a nuclear localisation (50). The exact reason for NBP35’s re-localisation remains unclear, but it may highlight the connection between the CIA and ISC pathways in apicomplexan parasites and reflects the complex evolutionary history of these proteins in eukaryotes (51–53)…”