Mitochondria have evolved diverse forms across eukaryotic diversity in adaptation to anoxia. Mitosomes are the simplest and the least well-studied type of anaerobic mitochondria. Transport of proteins via TIM complexes, composed of three proteins of the Tim17 protein family (Tim17/22/23), is one of the key unifying aspects of mitochondria and mitochondria-derived organelles. However, multiple experimental and bioinformatic attempts have so far failed to identify the nature of TIM in mitosomes of the anaerobic metamonad protist, Giardia intestinalis, one of the few experimental models for mitosome biology. Here, we present the identification of a single G. intestinalis Tim17 protein (GiTim17), made possible only by the implementation of a metamonad-specific hidden Markov model. While very divergent in primary sequence and in predicted membrane topology, experimental data suggest that GiTim17 is an inner membrane mitosomal protein, forming a disulphide-linked dimer. We suggest that the peculiar GiTim17 sequence reflects adaptation to the unusual, detergent resistant, inner mitosomal membrane. Specific pull-down experiments indicate interaction of GiTim17 with mitosomal Tim44, the tethering component of the import motor complex. Analysis of TIM complexes across eukaryote diversity suggests that a "single Tim" translocase is a convergent adaptation of mitosomes in anaerobic protists, with Tim22 and Tim17 (but not Tim23), providing the protein backbone.

Biology Centre CAS České Budějovice Czech Republic

Centre for Comparative Genomics and Evolutionary Bioinformatics Department of Biochemistry and Molecular Biology Dalhousie University Halifax Canada

Department of Genetics and Microbiology Charles University Praha 2 Czech Republic

Department of Parasitology Faculty of Science Charles University Vestec Czech Republic

Zobrazit více v PubMed

Alva V, Nam S-Z, Söding J, Lupas AN.. 2016. The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis. Nucleic Acids Res. 44(W1):W410–W415. PubMed PMC

Aurrecoechea C, et al. 2017. EuPathDB: the eukaryotic pathogen genomics database resource. Nucleic Acids Res. 45(D1):D581–D591. PubMed PMC

Burri L, Williams BAP, Bursac D, Lithgow T, Keeling PJ.. 2006. Microsporidian mitosomes retain elements of the general mitochondrial targeting system. Proc Natl Acad Sci U S A. 103(43):15916–15920. PubMed PMC

Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N.. 2009. Importing mitochondrial proteins: machineries and mechanisms. Cell 138(4):628–644. PubMed PMC

Collins LJ, Poole AM, Penny D.. 2003. Using ancestral sequences to uncover potential gene homologues. Appl Bioinformatics 2(3 Suppl):S85–S95. PubMed

Cox J, et al. 2014. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13(9):2513–2526. PubMed PMC

Dagley MJ, et al. 2009. The protein import channel in the outer mitosomal membrane of Giardia intestinalis. Mol Biol Evol. 26(9):1941–1947. PubMed PMC

Dawson SC, et al. 2007. Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis. Eukaryot Cell 6(12):2354–2364. PubMed PMC

Demishtein-Zohary K, et al. 2017. Role of Tim17 in coupling the import motor to the translocation channel of the mitochondrial presequence translocase. Elife 6:1–11. PubMed PMC

Dolezal P, et al. 2005. Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting. Proc Natl Acad Sci U S A. 102(31):10924–10929. PubMed PMC

Dolezal P, Likic V, Tachezy J, Lithgow T.. 2006. Evolution of the molecular machines for protein import into mitochondria. Science 313(5785):314–318. PubMed

Dudek J, Rehling P, van der Laan M.. 2013. Mitochondrial protein import: common principles and physiological networks. Biochim Biophys Acta-Mol Cell Res. 1833(2):274–285. PubMed

Eddy SR. 2011. Accelerated profile HMM searches. PLoS Comput Biol. 7(10):e1002195.. PubMed PMC

Fukasawa Y, Oda T, Tomii K, Imai K.. 2017. Origin and evolutionary alteration of the mitochondrial import system in eukaryotic lineages. Mol Biol Evol. 34(7):1574–1586. PubMed PMC

Garg S, et al. 2015. Conservation of transit peptide-independent protein import into the mitochondrial and hydrogenosomal matrix. Genome Biol Evol. 7(9):2716–2726. PubMed PMC

Gasteiger E, et al. 2005. Protein identification and analysis tools on the ExPASy server In: The proteomics protocols handbook. Totowa (NJ: ): Humana Press; p. 571–607.

Henriquez FL, Richards TA, Roberts F, McLeod R, Roberts CW.. 2005. The unusual mitochondrial compartment of Cryptosporidium parvum. Trends Parasitol. 21(2):68–74. PubMed

Hildebrand A, Remmert M, Biegert A, Soding J.. 2009. Fast and accurate automatic structure prediction with HHpred. Proteins 77 (Suppl 9):128–132. PubMed

Ishihama Y, Rappsilber J, Mann M.. 2006. Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J Proteome Res. 5(4):988–994. PubMed

Jakobs S, Wurm CA.. 2014. Super-resolution microscopy of mitochondria. Curr Opin Chem Biol. 20:9–15. PubMed

Jedelský PL, et al. 2011. The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis. PLoS ONE. 6(2):e17285. PubMed PMC

Käll L, Krogh A, Sonnhammer ELL.. 2007. Advantages of combined transmembrane topology and signal peptide prediction – the Phobius web server. Nucleic Acids Res. 35(Web Server):W429–W432. PubMed PMC

Katoh K, Standley DM.. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 30(4):772–780. PubMed PMC

Keister DB. 1983. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg. 77(4):487–488. PubMed

Kovermann P, et al. 2002. Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel. Mol Cell. 9(2):363–373. PubMed

Krogh A, Larsson B, von Heijne G, Sonnhammer EL.. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 305(3):567–580. PubMed

Kronidou NG, et al. 1994. Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. Proc Natl Acad Sci USA. 91(26):12818–12822. PubMed PMC

Leger MM, et al. 2017. Organelles that illuminate the origins of Trichomonas hydrogenosomes and Giardia mitosomes. Nat Ecol Evol. 1(4):0092. PubMed PMC

Leitsch D, Müller J, Müller N.. 2016. Evaluation of Giardia lamblia thioredoxin reductase as drug activating enzyme and as drug target. Int J Parasitol Drugs Drug Resist. 6(3):148–153. PubMed PMC

Likic VA, Dolezal P, Celik N, Dagley M, Lithgow T.. 2010. Using hidden markov models to discover new protein transport machines. Methods Mol Biol. 619:271–284. PubMed

Lithgow T, Schneider A.. 2010. Evolution of macromolecular import pathways in mitochondria, hydrogenosomes and mitosomes. Philos Trans R Soc Lond B Biol Sci. 365(1541):799–817. PubMed PMC

Martincová E, et al. 2012. Live imaging of mitosomes and hydrogenosomes by HaloTag technology. PLoS One. 7(4):e36314. PubMed PMC

Martincová E, et al. 2015. Probing the biology of Giardia intestinalis mitosomes using in vivo enzymatic tagging. Mol Cell Biol. 35(16):2864–2874. PubMed PMC

Martinez-Caballero S, Grigoriev SM, Herrmann JM, Campo ML, Kinnally KW.. 2007. Tim17p regulates the twin pore structure and voltage gating of the mitochondrial protein import complex TIM23. J Biol Chem. 282(6):3584–3593. PubMed

Masuda T, Tomita M, Ishihama Y.. 2008. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 7(2):731–740. PubMed

Mokranjac D, Neupert W.. 2010. The many faces of the mitochondrial TIM23 complex. Biochim Biophys Acta. 1797(6–7):1045–1054. PubMed

Morrison HG, et al. 2007. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317(5846):1921–1926. PubMed

Rehling P, et al. 2003. Protein insertion into the mitochondrial inner membrane by a twin-pore translocase. Science 299(5613):1747–1751. PubMed

Roger AJ, Muñoz-Gómez SA, Kamikawa R.. 2017. The origin and diversification of mitochondria. Curr Biol. 27(21):R1177–R1192. PubMed

Rout S, Zumthor JP, Schraner EM, Faso C, Hehl AB.. 2016. An interactome-centered protein discovery approach reveals novel components involved in mitosome function and homeostasis in Giardia lamblia. PLOS Pathog. 12(12):e1006036.. PubMed PMC

Schagger H, Pfeiffer K.. 2000. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19(8):1777–1783. PubMed PMC

Schneider A, Bursać D, Lithgow T.. 2008. The direct route: a simplified pathway for protein import into the mitochondrion of trypanosomes. Trends Cell Biol. 18(1):12–18. PubMed

Schneider H-C, et al. 1994. Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature 371(6500):768–774. PubMed

Shiflett AM, Johnson PJ.. 2010. Mitochondrion-related organelles in eukaryotic protists. Annu Rev Microbiol. 64:409–429. PubMed PMC

Singha UK, et al. 2008. Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei. Mol Biochem Parasitol. 159(1):30–43. PubMed PMC

Singha UK, et al. 2012. Protein translocase of mitochondrial inner membrane in Trypanosoma brucei. J Biol Chem. 287(18):14480–14493. PubMed PMC

Šmíd O, et al. 2008. Reductive evolution of the mitochondrial processing peptidases of the unicellular parasites Trichomonas vaginalis and Giardia intestinalis. PLoS Pathog. 4(12):e1000243. PubMed PMC

Sojo V, Dessimoz C, Pomiankowski A, Lane N.. 2016. Membrane proteins are dramatically less conserved than water-soluble proteins across the tree of life. Mol Biol Evol. 33(11):2874–2884. PubMed PMC

Ting S-Y, Yan NL, Schilke BA, Craig EA.. 2017. Dual interaction of scaffold protein Tim44 of mitochondrial import motor with channel-forming translocase subunit Tim23. Elife 6:1–22. PubMed PMC

Tovar J, et al. 2003. Mitochondrial remnant organelles of Giardia function in iron–sulphur protein maturation. Nature 426(6963):172–176. PubMed

Tyanova S, et al. 2016. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 13(9):731–740. PubMed

Vitali DG, et al. 2018. Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes. Elife 7:1–22. PubMed PMC

Voleman L, et al. 2017. Giardia intestinalis mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum. BMC Biol. 15:27. PubMed PMC

Žárský V, Doležal P.. 2016. Evolution of the Tim17 protein family. Biol Direct. 11(1):54.. PubMed PMC

Pam16 and Pam18 were repurposed during Trypanosoma brucei evolution to regulate the replication of mitochondrial DNA

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis

Genomics of Preaxostyla Flagellates Illuminates the Path Towards the Loss of Mitochondria

Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes

Adaptation of the late ISC pathway in the anaerobic mitochondrial organelles of Giardia intestinalis

Reduced mitochondria provide an essential function for the cytosolic methionine cycle

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates

High quality genome assembly of the amitochondriate eukaryote Monocercomonoides exilis

Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle

Retortamonads from vertebrate hosts share features of anaerobic metabolism and pre-adaptations to parasitism with diplomonads

The Mastigamoeba balamuthi Genome and the Nature of the Free-Living Ancestor of Entamoeba

Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system

The evolution of the Puf superfamily of proteins across the tree of eukaryotes

Mitochondrial dynamics in parasitic protists