Evolution of the Cytosolic Iron-Sulfur Cluster Assembly Machinery in Blastocystis Species and Other Microbial Eukaryotes

Abstract

bThe cytosolic iron/sulfur cluster assembly (CIA) machinery is responsible for the assembly of cytosolic and nuclear iron/sulfur clusters, cofactors that are vital for all living cells. This machinery is uniquely found in eukaryotes and consists of at least eight proteins in opisthokont lineages, such as animals and fungi. We sought to identify and characterize homologues of the CIA system proteins in the anaerobic stramenopile parasite Blastocystis sp. strain NandII. We identified transcripts encoding six of the components-Cia1, Cia2, MMS19, Nbp35, Nar1, and a putative Tah18 -and showed using immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation that the last three of them localized to the cytoplasm of the cell. We then used comparative genomic and phylogenetic approaches to investigate the evolutionary history of these proteins. While most Blastocystis homologues branch with their eukaryotic counterparts, the putative Blastocystis Tah18 seems to have a separate evolutionary origin and therefore possibly a different function. Furthermore, our phylogenomic analyses revealed that all eight CIA components described in opisthokonts originated before the diversification of extant eukaryotic lineages and were likely already present in the last eukaryotic common ancestor (LECA). The Nbp35, Nar1 Cia1, and Cia2 proteins have been conserved during the subsequent evolutionary diversification of eukaryotes and are present in virtually all extant lineages, whereas the other CIA proteins have patchy phylogenetic distributions. Cia2 appears to be homologous to SufT, a component of the prokaryotic sulfur utilization factors (SUF) system, making this the first reported evolutionary link between the CIA and any other Fe/S biogenesis pathway. All of our results suggest that the CIA machinery is an ubiquitous biosynthetic pathway in eukaryotes, but its apparent plasticity in composition raises questions regarding how it functions in nonmodel organisms and how it interfaces with various iron/sulfur cluster systems (i.e., the iron/sulfur cluster, nitrogen fixation, and/or SUF system) found in eukaryotic cells.

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

“…1i). Recent analyses of Dre2 from other eukaryotes suggested that it was present in the LECA54, but did not resolve its specific prokaryotic origin. Taken together, these data suggest that the CIA pathway is of mainly bacterial heritage and was already in place in the LECA.…”

The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron–sulfur proteins

“…Most of these components are essential for viability of yeast cells, and they are conserved in virtually all eukaryotes including human cells (Seki et al, 2013;Song and Lee, 2008;Stehling et al, 2008;Stehling et al, 2012;Tsaousis et al, 2014).…”

Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria

“…As a eukaryote, Blastocystis already contains the Cia1, Cia2, MMS19, Nbp35, Nar1, and Tah18 components of the cytoplasmic iron-sulfur cluster assembly (CIA) machinery, which is distinct from Suf. (Tsaousis et al , 2014) It also is puzzling that even though Bh -SufCB can enhance E. coli SufS and SufE activity, there are no homologues of SufS and SufE in Blastocystis . Therefore it is not clear how Bh -SufCB would obtain an Fe-S cluster in the cytosol unless it interacts with the CIA machinery.…”

“…(Wang et al , 2004) The sufT gene encodes a protein of unknown function although it is homologous to eukaryotic cytosolic and plastid Fe-S cluster biogenesis components (Cia2 and HCF101, respectively). (Schwenkert et al , 2010, Tsaousis et al , 2014) Attempts to delete the suf genes from M. smegmatis were unsuccessful, suggesting that the operon is essential in Mycobacteria . (Huet et al , 2005) Yeast two-hybrid assays using the M. tuberculosis genes revealed that SufB interacts with SufC and SufD in vivo.…”