Evolution of the Isd11/IscS complex reveals a single α- proteobacterial endosymbiosis for all eukaryotes

Transcription

1 MBE Advance Access published April 27, Letter to MBE Evolution of the Isd11/IscS complex reveals a single α- proteobacterial endosymbiosis for all eukaryotes Thomas A. Richards 1 and Mark van der Giezen 2 1 School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QG, UK 2 School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK Address for correspondence: Running title: Isd11 and the mitochondrial endosymbiosis Mark van der Giezen School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK Tel: Fax: m.vandergiezen@qmul.ac.uk Key words: mitochondria, mitosome, hydrogenosome, iron sulfur cluster, origin of the eukaryotic cell. Non standard abbreviations: HGT Horizontal Gene Transfer, MCMCMC -Metropoliscoupled Markov chain Monte Carlo, ISC Iron Sulfur Clusters. Title length: 103 characters Abstract length (in words): 136 words Total length of text, including all legends and methods, but not abstract (in characters including spaces): 2,189 words (15,568 characters incl. spaces) Total page requirement for all items (expressed as 0.7 pages, 0.5 pages, etc.): The Author Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org

2 2 Figure pages Figure pages Total references: 22 Supplementary methods 852 words (6,342 characters incl. spaces) 9 References Supplementary Figure pages Supplementary Table 1. 2 pages Giardia and Trichomonas are eukaryotes without standard mitochondria but contain mitochondrial-type α-proteobacterium-derived iron-sulfur cluster (ISC) assembly proteins, located to mitosomes in Giardia and hydrogenosomes in Trichomonas. Although these data suggest a single common endosymbiotic ancestry for mitochondria, mitosomes and hydrogenosomes, separate origins are still being proposed. Here, we present a bioinformatic analysis of Isd11, a recently described essential component of the mitochondrial ISC assembly pathway. Isd11 is unique to eukaryotes but functions closely with the α-proteobacterium-derived cysteine desulfurase IscS. We demonstrate the presence of homologues of Isd11 in all five eukaryotic supergroups sampled, including hydrogenosomal and mitosomal lineages. The eukaryotic invention of Isd11 as a functional partner to IscS, directly implies a single shared α-proteobacterial endosymbiotic ancestry for all eukaryotes. This pinpoints the α-proteobacterial endosymbiosis to before the last common ancestor of all eukaryotes without ambiguity. Endosymbiosis played a key role in the evolution of eukaryotic cells but the number and ancestry of endosymbiotic events remains contentious (Martin and Müller 1998; Martin et al. 2001; Dyall, Brown, and Johnson 2004). Did organelles such as hydrogenosomes, mitosomes and mitochondria originate separately from different symbiotic bacteria, or

3 3 from just one endosymbiosis (Dyall, Brown, and Johnson 2004; Embley and Martin 2006)? In many species, mitochondria are essential for energy transduction by oxidative phosphorylation. However, systematic deletion of mitochondrial proteins has demonstrated that yeast mitochondria perform only one essential function; the assembly of iron-sulfur clusters (Lill and Mühlenhoff 2005). This suggests that mitochondria are retained for compartmentalized ISC assembly (Lill and Mühlenhoff 2005; van der Giezen and Tovar 2005). Wiedemann et al. (2006) and Adam et al. (2006) recently demonstrated that the ISC protein Isd11 is localized within mitochondria and is essential for ISC biogenesis in yeast and that Isd11 forms a complex with Nfs1. Nfs1 and its orthologue IscS, play a role as a vital cysteine desulfurase of the ISC assembly machinery (Adam et al. 2006; Wiedemann et al. 2006). Isd11 is suggested to function as an adapter and stabilizer of Nfs1 (Adam et al. 2006; Wiedemann et al. 2006). Homologues of the Isd11 gene have been detected in plant, fungi and animal genomes, which possess mitochondria, but no prokaryote homologue has been found (Wiedemann et al. 2006). This suggests Isd11 is a eukaryotic molecular innovation that arose, at the earliest, during the primary process of endosymbiosis that gave rise to mitochondria and which installed IscS, the functional partner of Isd11 (Adam et al. 2006; Wiedemann et al. 2006), in the eukaryotes. To test this hypothesis, we searched for putative homologues from available prokaryotic genomes using multiple PSI-BLAST searches of GenBank and confirmed, given current sampling, that the Isd11 gene is exclusive to eukaryotes. BLAST searches of all available eukaryotic genomes revealed the presence of putative IscS homologues in all eukaryotes surveyed, except for incomplete genome projects (Figure 1A). Further comparative genome analyses detected putative homologues of Isd11 in numerous eukaryotes including hydrogenosomal and mitosomal lineages. BLASTp analyses

4 4 demonstrated 47% amino acid sequence identity with the yeast Isd11 protein for both the Trichomonas vaginalis and Nosema locustae (synonym Antonospora locustae) putative Isd11 proteins. Together with the protein alignment this suggests that the amitochondrial sequences are true homologues (Figure 1B). Isd11 has a high helical propensity (see Figure 1B). Interestingly, neither PSSM (Kelley, MacCallum, and Sternberg 2000) nor the newer PHYRE algorithm ( recognise any known fold in Isd11, suggesting Isd11 might represent a new type of protein fold. Further structural studies, especially in complex with Nfs1, should prove interesting. All the putative Isd11 homologues analysed are predicted to contain three α- helices, consistent with their shared homology. Bioinformatic predictions of mitochondrial targeting for Isd11 were unconvincing for all lineages investigated, including the yeast Isd11, known to be located in mitochondria (Adam et al. 2006; Wiedemann et al. 2006) but demonstrated a candidate target peptide in the putative Isd11 of T. vaginalis (Figure 1B and Supplementary Table 1). The process of horizontal gene transfers (HGT) could theoretically distribute genes of endosymbiotic ancestry into lineages that did not undergo the original endosymbiosis (Roger et al. 1998). The Isd11 gene is short (encoding ~90 amino acids) and unlikely to be a reliable gene for phylogeny, indeed like many single-gene eukaryote phylogenies, the terminal branches were unresolved (Figure 2A see branches within the grey zone). Comparison tests of alternative terminal branching orders confirmed that many of the pictured terminal node relationships are unresolved (see methods and Supplementary Figure 1). However, phylogenetic analysis did not provide strong support for HGT over the more parsimonious scenario of vertical inheritance for the T. vaginalis Isd11 gene (Figure 2A). Furthermore, Nosema Isd11 consistently grouped within a weakly resolved clade of alveolates and formed a moderately supported sister group relationship with the Plasmodium parasites in all analyses. Plasmodium and Microsporidia are intracellular parasites that infect insects,

5 5 and in both Plasmodium and Microsporidia there are reported cases of HGT between these parasites and cells that they come into close contact with (Richards et al. 2003). Comparative topology tests could not reject an alternative topology where the Microsporidia branched below the alveolate group (for example approximately unbiased [AU] test - P = 0.49 (Shimodaira and Hasegawa 2001)), which is inconsistent with a Plasmodium-to-Microsporidia gene transfer. However, taken together, we cannot exclude a case of HGT between the Apicomplexa (Plasmodium) and Microsporidia (Nosema) based on the Isd11 phylogeny but this scenario is less parsimonious than vertical inheritance. In conclusion, we suggest that Isd11 originated during, or shortly after, the single endosymbiosis that gave rise to mitochondria, hydrogenosomes and mitosomes. Isd11 evolved as an exclusively eukaryotic addition to the α-proteobacterium-derived ISC biogenesis pathway. Like the ISC biogenesis pathway itself, Isd11 has been conserved in hydrogenosomal and mitosomal lineages. We demonstrate that Isd11 represents a unique shared derived character of all sampled eukaryote super-groups (Figure 2B), including mitochondrial, hydrogenosomal and mitosomal lineages. Isd11 therefore pinpoints the ancestry of the eukaryotic ISC biogenesis pathway to a single endosymbiotic event. The alternative assumption of separate origins of the IscS/Isd11 complex requires Isd11 to have evolved convergently in separate lineages and IscS to be acquired separately from two closely related proteobacteria, while separate endosymbiotic origins for any combination of the three organelles must include a process of endosymbiotic replacement (Dyall, Brown, and Johnson 2004), these alternative scenarios may be possible but are very unparsimonious. Similarities in mitochondrial, hydrogenosomal and mitosomal N-terminal targeting peptides also hint at a shared derived import machinery in all three organelle types suggesting a common origin of these organelles (van der Giezen and Tovar 2005). However, although genes encoding components of a mitochondrial import machinery have been identified in

6 6 mitosomal lineages (Henriquez et al. 2005) no such genes have been identified in hydrogenosomal lineages yet. In conclusion, the IscS/Isd11 complex was present in the last common ancestor of all the eukaryotes, prior to the division of all eukaryotic supergroups (Simpson and Roger 2004). Therefore, the α-proteobacterial endosymbiosis is placed firmly before the radiation of all eukaryotes (Figure 2B). Supplementary Materials Additional detailed methods and results of alternative topology comparison tests (Supplementary Figure 1) and comparisons of putative Isd11 mitochondria targeting peptides (Supplementary Table 1) are available at Molecular Biology and Evolution online ( Acknowledgments We are grateful to TIGR ( The Department of Energy ( Genoscope ( and MBL Woods Hole ( for making their genome data available (01/06). TAR thanks DEFRA for fellowship support. We thank NJ Talbot and JF Allen for comments and P Foster with assistance with topology comparison tests. Literature Cited Adam, A. C., C. Bornhövd, H. Prokisch, W. Neupert, and K. Hell The Nfs1 interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in mitochondria. EMBO J. 25: Andersson, J. O., S. W. Sarchfield, and A. J. Roger Gene transfers from nanoarchaeota to an ancestor of diplomonads and parabasalids. Mol. Biol. Evol. 22: Cavalier-Smith, T. 2003a. The excavate protozoan phyla Metamonada Grassé emend. (Anaeromonadea, Parabasalia, Carpediemonas, Eopharyngia) and Loukozoa emend. (Jakobea, Malawimonas): their evolutionary affinities and new higher taxa. Int. J. Syst. Evol. Microbiol. 53:

7 7 Cavalier-Smith, T. 2003b. Protist phylogeny and the high-level classification of protozoa. Eur. J. Protistol. 39: Dyall, S. D., M. T. Brown, and P. J. Johnson Ancient invasions: from endosymbionts to organelles. Science 304: Embley, T. M., and W. Martin Eukaryotic evolution, changes and challenges. Nature 440: Henriquez, F. L., T. A. Richards, F. Roberts, R. McLeod, and C. W. Roberts The unusual mitochondrial compartment of Cryptosporidium parvum. Trends Parasitol. 21: Kelley, L. A., R. M. MacCallum, and M. J. Sternberg Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299: Lill, R., and U. Mühlenhoff Iron-sulfur-protein biogenesis in eukaryotes. Trends Biochem Sci 30: Martin, W., M. Hoffmeister, C. Rotte, and K. Henze An overview of endosymbiotic models for the origins of eukaryotes, their ATP-producing organelles (mitochondria and hydrogenosomes), and their heterotrophic lifestyle. Biol Chem 382: Martin, W., and M. Müller The hydrogen hypothesis for the first eukaryote. Nature 392: Richards, T. A., and T. Cavalier-Smith Myosin domain evolution and the primary divergence of eukaryotes. Nature 436: Richards, T. A., R. P. Hirt, B. A. Williams, and T. M. Embley Horizontal gene transfer and the evolution of parasitic protozoa. Protist 154: Rodriguez-Ezpeleta, N., H. Brinkmann, S. C. Burey, B. Roure, G. Burger, W. Loffelhardt, H. J. Bohnert, H. Philippe, and B. F. Lang Monophyly of primary photosynthetic eukaryotes: green plants, red algae, and glaucophytes. Curr. Biol. 15: Roger, A. J., S. G. Svard, J. Tovar, C. G. Clark, M. W. Smith, F. D. Gillin, and M. L. Sogin A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria. Proc. Natl. Acad. Sci. USA 95: Shimodaira, H., and M. Hasegawa CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17: Simpson, A. G Cytoskeletal organization, phylogenetic affinities and systematics in the contentious taxon Excavata (Eukaryota). Int. J. Syst. Evol. Microbiol. 53: Simpson, A. G., and A. J. Roger The real 'kingdoms' of eukaryotes. Curr. Biol. 14:R Stechmann, A., and T. Cavalier-Smith Rooting the eukaryote tree by using a derived gene fusion. Science 297: van der Giezen, M., S. Cox, and J. Tovar The iron-sulfur cluster assembly genes iscs and iscu of Entamoeba histolytica were acquired by horizontal gene transfer. BMC Evol. Biol. 4:7. van der Giezen, M., and J. Tovar Degenerate mitochondria. EMBO Rep 6:

8 8 Wiedemann, N., E. Urzica, B. Guiard, H. Muller, C. Lohaus, H. E. Meyer, M. T. Ryan, C. Meisinger, U. Muhlenhoff, R. Lill, and N. Pfanner Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins. EMBO J. 25: Figure 1. (A) Eukaryotic genome survey for putative homologues of IscS and Ids11. Yellow indicates putative homologue present. Black indicates absence of similar sequences. Red indicates evidence of HGT (van der Giezen, Cox, and Tovar 2004) possibly explaining absence of the Isd11/IscS complex by functional replacement with a eubacterial IscS. Triangles indicate amitochondrial lineages. (B) Alignment of a taxonomically diverse representation of putative Isd11 proteins. Residues with 50% conservation are shaded black. The LYR/K residue block (Pfam PF05347) conserved in Isd11 and B14 and B22 components of mitochondrial complex I are illustrated, note that all sampled Excavata possess a tyrosine deletion and that sequence similarity between the Isd11 and B14 and B22 is negligible beyond the LYR/K block. Circles below the alignment represent positions conserved in 50% or more of the sequences and present in Trichomonas (Green) or Nosema (Orange). The purple peptide model shows the predicted consensus structural arrangement of α-helices. Purple alignment shading indicates conformity with predicted helical regions. Predicted amphipathic α-helix of the putative Trichomonas hydrogenosomal targeting sequence is shaded purple with a red border (see Supplementary Table 1 for details about putative N-terminal target peptide detection). Figure 2. (A) Phylogeny of Isd11 calculated from an alignment of 23 taxa and 58 conserved amino acid alignment characters. Topology is shown unrooted. Topology support values are shown when PHYML bootstrap values are in excess of 50%. Support values are shown in the order 1) Bayesian posterior probability 2) % bootstrap support from 1,000 PHYML bootstrap replicates and 3) % bootstrap support from 1,000 PROTPARS bootstrap replicates. Eukaryote supergroup classifications are notated next to species names. An alternative branching order, demonstrating alveolate monophyly

9 9 and tested using CONSEL (see Supplementary Methods) is illustrated with a grey branch. The grey zone indicates tree nodes unresolved in all analyses (i.e. all topology support values below 0.5 posterior probability and 50% bootstrap support). GenBank accession numbers for sequences used in phylogeny: At, AAM66015; Os, NP_921405; Cm, CMN136C ( Lm, CAJ06332, Tc, XP_807608; Tb, XP_823406; Sc, NP_010968; Kl, XP_454147; Um, EAK83440, Dp, EAL31501; Rn, XP_574009; Dr, XP_701063; Dd, XP_635573; Pf, CAD52245; Pb, XP_ All other sequences were predicted open reading frames from genome projects as discussed in the Supplementary Methods. (B) Pinpointing the evolution of the Isd11/IscS complex within the eukaryotes. Note, that the bikont/unikont root model is used (Stechmann and Cavalier-Smith 2002; Richards and Cavalier-Smith 2005). The Isd11 tyrosine deletion (Figure 1B), strong results of multi-gene phylogeny (Rodriguez-Ezpeleta et al. 2005) and morphological characters suggest Excavata monophyly (Cavalier-Smith 2003a; Simpson 2003) while bikont monophyly is supported by the dhfr-ts fusion character (Stechmann and Cavalier-Smith 2002), inferred ancestral morphological characters (Stechmann and Cavalier-Smith 2002; Cavalier-Smith 2003b) and results of unrooted multi-gene phylogenies (Rodriguez-Ezpeleta et al. 2005). This data, coupled with shared derived HGTs present in Trichomonas and Giardia (Andersson, Sarchfield, and Roger 2005) favour the tree topology shown. The alternative Giardia/Trichomonas root requires fission events in the ancestral unikont dhfr-ts and separate origins or separate evolutionary reductions of highly complex morphological characters and the presence of such characters in the last common eukaryotic ancestor (Cavalier-Smith 2003a; Simpson 2003). However, this alternative root would still pinpoint the Isd11/IscS character to the base of the eukaryote radiation, demonstrating that the α-proteobacterial endosymbiosis giving rise to mitochondria, hydrogenosomes and mitosomes occurred in the last common eukaryotic ancestor.

10

11 + A. B. Chromalveolates (Diatom) nikonts (Fungi) Unikonts Microsporidia) 0.1 Subs/site Thalassiosira pseudonana Ustilago maydis Phanerochaete chrysosporium Kluyveromyces lactis 0.98/75/66 Saccharomyces 0.81/50/41 cerevisiae Nosema locustae Unikonts (Amoebozoa) Plasmodium falciparum Plasmodium berghei Chromalveolates (Apicomplexa) Dictyostelium discoideum Rhizopus oryzae 1/100/100 Drosophila pseudoobscura Danio rerio Unikonts (Metazoa) 0.71/68 / /61/66 1/86/74 Paramecium tetraurelia Rattus norvegicus 0.99/62/ /76/44 Tetrahymena thermophila Chromalveolates (Ciliates) Cyanidioschyzon merolae Oryza sativa 1/100/99 Chlamydomonas reinhardtii Arabidopsis thaliana Leishmania major 0.98/81/62 Trichomonas vaginalis Trypanosoma cruzi Trypanosoma brucei Plantae Excavata Excavata (Kinetoplastids) DHFR-TS Fusion LYR/K Tyrosine deletion DHFR-TS loss IscS Isd11 endosymbiotic transmission Excavata (Trichomonas & Giardia) Excavata (Kinetoplastids) Plantae Chromalveolates Unikonts Prokaryotes Richards and van der Giezen Fig. 2