The origin of protein import was a key step in the endosymbiotic acquisition of mitochondria. Though the main translocon of the mitochondrial outer membrane, TOM40, is ubiquitous among organelles of mitochondrial ancestry, the transit peptides, or N-terminal targeting sequences (NTSs), recognised by the TOM complex, are not. To better understand the nature of evolutionary conservation in mitochondrial protein import, we investigated the targeting behavior of Trichomonas vaginalis hydrogenosomal proteins in Saccharomyces cerevisiae and vice versa. Hydrogenosomes import yeast mitochondrial proteins even in the absence of their native NTSs, but do not import yeast cytosolic proteins. Conversely, yeast mitochondria import hydrogenosomal proteins with and without their short NTSs. Conservation of an NTS-independent mitochondrial import route from excavates to opisthokonts indicates its presence in the eukaryote common ancestor. Mitochondrial protein import is known to entail electrophoresis of positively charged NTSs across the electrochemical gradient of the inner mitochondrial membrane. Our present findings indicate that mitochondrial transit peptides, which readily arise from random sequences, were initially selected as a signal for charge-dependent protein targeting specifically to the mitochondrial matrix. Evolutionary loss of the electron transport chain in hydrogenosomes and mitosomes lifted the selective constraints that maintain positive charge in NTSs, allowing first the NTS charge, and subsequently the NTS itself, to be lost. This resulted in NTS-independent matrix targeting, which is conserved across the evolutionary divide separating trichomonads and yeast, and which we propose is the ancestral state of mitochondrial protein import.

Department of Parasitology Charles University Prague Faculty of Science Czech Republic

Institute for Molecular Evolution Heinrich Heine University Düsseldorf Düsseldorf Germany

Zobrazit více v PubMed

Aurrecoechea C, et al. 2008. GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis. Nucleic Acids Res. 37:D526–D530. PubMed PMC

Baker A, Schatz G. 1987. Sequences from a prokaryotic genome or the mouse dihydrofolate reductase gene can restore the import of a truncated precursor protein into yeast mitochondria. Proc Natl Acad Sci U S A. 84:3117–3121. PubMed PMC

Burstein D, et al. 2012. A machine learning approach to identify hydrogenosomal proteins in Trichomonas vaginalis. Eukaryot Cell. 11:217–228. PubMed PMC

Carlton JM, et al. 2007. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315:207–212. PubMed PMC

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

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

Donahue SL. 2001. Mitochondrial DNA ligase function in Saccharomyces cerevisiae. Nucleic Acids Res. 29:1582–1589. PubMed PMC

Emanuelsson O, Nielsen H, Brunak S, von Heijne G. 2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol. 300:1005–1016. PubMed

Endo T, Yamano K. 2009. Transport of proteins across or into the mitochondrial outer membrane. Biochim Biophys Acta. 1803:706–714. PubMed

Esaki M, et al. 2004. Mitochondrial protein import. J Biol Chem. 279:45701–45707. PubMed

Geissler A, et al. 2000. Membrane potential-driven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence. Mol Biol Cell. 11:3977–3991. PubMed PMC

Goldberg AV, et al. 2008. Localization and functionality of microsporidian iron–sulphur cluster assembly proteins. Nature 452:624–628. PubMed

Gorrell TE, Yarlett N, Müller M. 1984. Isolation and characterization of Trichomonas vaginalis ferredoxin. Carlsberg Res Commun. 49:259–268.

Gregg C, Kyryakov P, Titorenko VI. 2009. Purification of mitochondria from yeast cells. J Vis Exp. 24. PubMed PMC

Hamilton V, Singha UK, Smith JT, Weems E, Chaudhuri M. 2014. Trypanosome alternative oxidase possesses both an N-terminal and internal mitochondrial targeting signal. Eukaryot Cell. 13:539–547. PubMed PMC

Häusler T, Stierhof YD, Blattner J, Clayton C. 1997. Conservation of mitochondrial targeting sequence function in mitochondrial and hydrogenosomal proteins from the early-branching eukaryotes Crithidia, Trypanosoma and Trichomonas. Eur J Cell Biol. 73:240–251. PubMed

Heinz E, Lithgow T. 2013. Back to basics: a revealing secondary reduction of the mitochondrial protein import pathway in diverse intracellular parasites. Biochim Biophys Acta. 1833:295–303. PubMed

Hewitt V, Alcock F, Lithgow T. 2011. Minor modifications and major adaptations: the evolution of molecular machines driving mitochondrial protein import. Biochim Biophys Acta. 1808:947–954. PubMed

Hewitt VL, et al. 2012. A model system for mitochondrial biogenesis reveals evolutionary rewiring of protein import and membrane assembly pathways. Proc Natl Acad Sci U S A. 109:E3358–E3366. PubMed PMC

Jedelský PL, et al. 2010. The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis. PLoS One 6:e17285–e17285. PubMed PMC

Katinka MD, et al. 2001. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature 414:450–453. PubMed

Kusdian G, Woehle C, Martin WF, Gould SB. 2013. The actin-based machinery of Trichomonas vaginalis mediates flagellate-amoeboid transition and migration across host tissue. Cell Microbiol. 15:1707–1721. PubMed

Land KM, et al. 2003. Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance. Mol Microbiol. 51:115–122. PubMed

Lane N, Martin W. 2010. The energetics of genome complexity. Nature 467:929–934. PubMed

Lill R, Neupert W. 1996. Mechanisms of protein import across the mitochondrial outer membrane. Trends Cell Biol. 6:56–61. PubMed

Lindmark DG, Muller M. 1973. Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism. J Biol Chem. 248:7724–7728. PubMed

Lucattini R, Likic VA, Lithgow T. 2004. Bacterial proteins predisposed for targeting to mitochondria. Mol Biol Evol. 21:652–658. PubMed

Makiuchi T, Nozaki T. 2014. Highly divergent mitochondrion-related organelles in anaerobic parasitic protozoa. Biochimie. 100:3–17. PubMed

Martin J, Mahlke K, Pfanner N. 1991. Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences. J Biol Chem. 266:18051–18057. PubMed

Martin W, Koonin EV. 2006. Introns and the origin of nucleus—cytosol compartmentalization. Nature 440:41–45. PubMed

McInerney JO, O'Connell MJ, Pisani D. 2014. The hybrid nature of the Eukaryota and a consilient view of life on Earth. Nat Rev Microbiol. 12:449–455. PubMed

Meisinger C, Sickmann A, Pfanner N. 2008. The mitochondrial proteome: from inventory to function. Cell 134:22–24. PubMed

Mentel M, Zimorski V, Haferkamp P, Martin W, Henze K. 2008. Protein import into hydrogenosomes of Trichomonas vaginalis involves both N-terminal and internal targeting signals: a case study of thioredoxin reductases. Eukaryot Cell. 7:1750–1757. PubMed PMC

Mi-ichi F, Abu Yousuf M, Nakada-Tsukui K, Nozaki T. 2009. Mitosomes in Entamoeba histolytica contain a sulfate activation pathway. Proc Natl Acad Sci U S A. 106:21731–21736. PubMed PMC

Muller M. 1993. The hydrogenosome. J Gen Microbiol. 139:2879–2889. PubMed

Muller M, et al. 2012. Biochemistry and evolution of anaerobic energy metabolism in eukaryotes. Microbiol Mol Biol Rev. 76:444–495. PubMed PMC

Neupert W. 2015. A perspective on transport of proteins into mitochondria: a myriad of open questions. J Mol Biol. 427:1135–1158. PubMed

Neupert W, Herrmann JM. 2007. Translocation of proteins into mitochondria. Annu Rev Biochem. 76:723–749. PubMed

Noël CJ, et al. 2010. Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics. BMC Genomics 11:99–125. PubMed PMC

Pérez JMM, Pascau J. 2013. Image processing with ImageJ. Packt Publishing Ltd.

Perry AJ, Hulett JM, Likic VA, Lithgow T, Gooley PR. 2006. Convergent evolution of receptors for protein import into mitochondria. Curr Biol. 16:221–229. PubMed

Pfanner N, Neupert W. 1986. Transport of F1-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett. 209:152–156. PubMed

Rada P, et al. 2011. The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis. PLoS One 6:e24428. PubMed PMC

Rassow J, Dekker PJT, van Wilpe S, Meijer M, Soll J. 1999. The preprotein translocase of the mitochondrial inner membrane: function and evolution. J Mol Biol. 286:105–120. PubMed

Regoes A, et al. 2005. Protein import, replication, and inheritance of a vestigial mitochondrion. J Biol Chem. 280:30557–30563. PubMed

Rice P, Longden I, Bleasby A. 2000. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16:276–277. PubMed

Schleiff EE, Becker TT. 2010. Common ground for protein translocation: access control for mitochondria and chloroplasts. Nat Rev Mol Cell Biol. 12:48–59. PubMed

Schneider RE, et al. 2011. The Trichomonas vaginalis hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes. Int J Parasitol. 41:1421–1434. 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:e1000243. PubMed PMC

Timmis JN, Ayliffe MA, Huang CY, Martin W. 2004. Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet. 5:123–135. PubMed

Todisco S, et al. 2014. The Saccharomyces cerevisiae gene YPR011c encodes a mitochondrial transporter of adenosine 5′-phosphosulfate and 3′-phospho-adenosine 5′-phosphosulfate. Biochim Biophys Acta. 1837:326–334. PubMed

Van der Giezen M, et al. 2002. Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles. EMBO J. 21:572–579. PubMed PMC

Vögtle FN, et al. 2009. Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability. Cell 139:428–439. PubMed

von Heijne G. 1986. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5:1335–1471. PubMed PMC

Waller RF, et al. 2009. Evidence of a reduced and modified mitochondrial protein import apparatus in microsporidian mitosomes. Eukaryot Cell. 8:19–26. PubMed PMC

Williams TA, Foster PG, Cox CJ, Embley TM. 2013. An archaeal origin of eukaryotes supports only two primary domains of life. Nature 504:231–236. PubMed

Zeth K, Thein M. 2010. Porins in prokaryotes and eukaryotes: common themes and variations. Biochem J. 431:13–22. PubMed

Zimorski V, et al. 2013. The N-terminal sequences of four major hydrogenosomal proteins are not essential for import into hydrogenosomes of Trichomonas vaginalis. J Eukaryot Microbiol. 60:89–97. PubMed

Zubácová Z, Hostomská J, Tachezy J. 2012. Histone H3 variants in Trichomonas vaginalis. Eukaryot Cell. 11:654–661 PubMed PMC

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

Comparative analysis of mitochondrion-related organelles in anaerobic amoebozoans

A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle

Reduced mitochondria provide an essential function for the cytosolic methionine cycle

Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics

Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis

A Single Tim Translocase in the Mitosomes of Giardia intestinalis Illustrates Convergence of Protein Import Machines in Anaerobic Eukaryotes

Mitochondrial Glycolysis in a Major Lineage of Eukaryotes

Fe-S cluster assembly in the supergroup Excavata

Organelles that illuminate the origins of Trichomonas hydrogenosomes and Giardia mitosomes

A Comparative Analysis of Mitochondrial Genomes in Eustigmatophyte Algae

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis