The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.

Department of Parasitology Faculty of Science Charles University Prague Prague Czech Republic

Zobrazit více v PubMed

Andersson SG, Zomorodipour A, Andersson JO, Sicheritz-Ponten T, Alsmark UC, et al. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature. 1998;396:133–140. PubMed

Lang BF, Burger G, O'Kelly CJ, Cedergren R, Golding GB, et al. An ancestral mitochondrial DNA resembling a eubacterial genome in miniature. Nature. 1997;387:493–497. PubMed

Vaidya AB, Mather MW. Mitochondrial evolution and functions in malaria parasites. Annu Rev Microbiol. 2009;63:249–267. 10.1146/annurev.micro.091208.073424 [doi] PubMed

Gabaldon T, Huynen MA. Shaping the mitochondrial proteome. Biochim Biophys Acta. 2004;1659:212–220. PubMed

Gabaldon T, Huynen MA. From endosymbiont to host-controlled organelle: the hijacking of mitochondrial protein synthesis and metabolism. PLoS Comput Biol. 2007;3:e219. PubMed PMC

Pagliarini DJ, Calvo SE, Chang B, Sheth SA, Vafai SB, et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell. 2008;134:112–123. PubMed PMC

Tachezy J, Smid O. Mitosomes in parasitic protists. In: Tachezy J, editor. Hydrogenosomes and Mitosomes: Mitochondria of Anaerobic Eukaryotes. Berlin, Heidelberg: Springer-Verlag; 2008. pp. 201–230.

Cavalier-Smith T. The origin of eukaryotic and archaebacterial cells. Ann N Y Acad Sci. 1987;503:17–54:17–54. PubMed

Tovar J, Leon-Avila G, Sanchez LB, Sutak R, Tachezy J, et al. Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature. 2003;426:172–176. PubMed

Tovar J, Fischer A, Clark CG. The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica. Mol Microbiol. 1999;32:1013–1021. PubMed

Katinka MD, Duprat S, Cornillot E, Metenier G, Thomarat F, et al. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature. 2001;414:450–453. PubMed

Williams BAP, Hirt RP, Lucocq JM, Embley TM. A mitochondrial remnant in the microsporidian Trachipleistophora hominis. Nature. 2002;418:865–869. PubMed

Riordan CE, Langreth SG, Sanchez LB, Kayser O, Keithly JS. Preliminary evidence for a mitochondrion in Cryptosporidium parvum: Phylogenetic and therapeutic implications. Journal of Eukaryotic Microbiology. 1999;46:52S–55S. PubMed

Hrdy I, Tachezy J, Müller M. Metabolism of trichomonad hydrogenosomes. In: Tachezy J, editor. Hydrogenosomes and Mitosomes:Mitochondria of Anaerobic Euakryotes. Berlin, Heidelberg: Springer-Verlag; 2008. pp. 114–145.

Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, et al. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science. 2004;304:441–445. 10.1126/science.1094786 [doi];1094786 [pii] PubMed

Dolezal P, Smid O, Rada P, Zubacova Z, Bursac D, et al. Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting. Proc Natl Acad Sci U S A. 2005;102:10924–10929. PubMed PMC

Goldberg AV, Molik S, Tsaousis AD, Neumann K, Kuhnke G, et al. Localization and functionality of microsporidian iron-sulphur cluster assembly proteins. Nature. 2008;452:624–628. PubMed

Putignani L, Tait A, Smith HV, Horner D, Tovar J, et al. Characterization of a mitochondrion-like organelle in Cryptosporidium parvum. Parasitology. 2004;129:1–18. PubMed

Sanderson SJ, Xia D, Prieto H, Yates J, Heiges M, et al. Determining the protein repertoire of Cryptosporidium parvum sporozoites. Proteomics. 2008;8:1398–1414. PubMed PMC

Tsaousis AD, Kunji ER, Goldberg AV, Lucocq JM, Hirt RP, et al. A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi. Nature. 2008;453:553–556. nature06903 [pii];10.1038/nature06903 [doi] PubMed

Xu P, Widmer G, Wang Y, Ozaki LS, Alves JM, et al. The genome of Cryptosporidium hominis. Nature. 2004;431:1107–1112. PubMed

Mi-ichi F, Abu YM, Nakada-Tsukui K, Nozaki T. Mitosomes in Entamoeba histolytica contain a sulfate activation pathway. Proc Natl Acad Sci U S A. 2009;106:21731–21736. 0907106106 [pii];10.1073/pnas.0907106106 [doi] PubMed PMC

Franzen O, Jerlstrom-Hultqvist J, Castro E, Sherwood E, Ankarklev J, et al. Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species? PLoS Pathog. 2009;5:e1000560. 10.1371/journal.ppat.1000560 [doi] PubMed PMC

Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, et al. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science. 2007;317:1921–1926. PubMed

Cavalier-Smith T. Kingdoms Protozoa and Chromista and the eozoan root of the eukaryotic tree. Biol Lett. 2010;6:342–345. PubMed PMC

Billington D, Maltby PJ, Jackson AP, Graham JM. Dissection of hepatic receptor-mediated endocytic pathways using self-generated gradients of iodixanol (Optiprep). Anal Biochem. 1998;258:251–258. S0003-2697(98)92561-1 [pii];10.1006/abio.1998.2561 [doi] PubMed

Dunkley TP, Watson R, Griffin JL, Dupree P, Lilley KS. Localization of organelle proteins by isotope tagging (LOPIT). Mol Cell Proteomics. 2004;3:1128–1134. 10.1074/mcp.T400009-MCP200 [doi];T400009-MCP200 [pii] PubMed

Smid O, Matuskova A, Harris SR, Kucera T, Novotny M, et al. Reductive evolution of the mitochondrial processing peptidases of the unicellular parasites Trichomonas vaginalis and Giardia intestinalis. PLoS Pathog. 2008;4:e1000243. PubMed PMC

Rada P, Smid O, Sutak R, Dolezal P, Pyrih J, et al. The monothiol single-domain glutaredoxin is conserved in the highly reduced mitochondria of Giardia intestinalis. Eukaryot Cell. 2009;8:1584–1591. EC.00181-09 [pii];10.1128/EC.00181-09 [doi] PubMed PMC

Tachezy J, Sanchez LB, Müller M. Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS. Molecular Biology and Evolution. 2001;18:1919–1928. PubMed

Vinella D, Brochier-Armanet C, Loiseau L, Talla E, Barras F. Iron-sulfur (Fe/S) protein biogenesis: phylogenomic and genetic studies of A-type carriers. PLoS Genet. 2009;5:e1000497. 10.1371/journal.pgen.1000497 [doi] PubMed PMC

Krebs C, Agar JN, Smith AD, Frazzon J, Dean DR, et al. IscA, an alternate scaffold for Fe-S cluster biosynthesis. Biochemistry. 2001;40:14069–14080. bi015656z [pii] PubMed

Pelzer W, Muhlenhoff U, Diekert K, Siegmund K, Kispal G, et al. Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron-sulfur proteins. FEBS Lett. 2000;476:134–139. S0014-5793(00)01711-7 [pii] PubMed

Song D, Tu Z, Lee FS. Human ISCA1 interacts with IOP1/NARFL and functions in both cytosolic and mitochondrial iron-sulfur protein biogenesis. J Biol Chem. 2009;284:35297–35307. M109.040014 [pii];10.1074/jbc.M109.040014 [doi] PubMed PMC

Ding H, Clark RJ, Ding B. IscA mediates iron delivery for assembly of iron-sulfur clusters in IscU under the limited accessible free iron conditions. J Biol Chem. 2004;279:37499–37504. 10.1074/jbc.M404533200 [doi];M404533200 [pii] PubMed

Bych K, Kerscher S, Netz DJ, Pierik AJ, Zwicker K, et al. The iron-sulphur protein Ind1 is required for effective complex I assembly. EMBO J. 2008;27:1736–1746. emboj200898 [pii];10.1038/emboj.2008.98 [doi] PubMed PMC

Sheftel AD, Stehling O, Pierik AJ, Netz DJ, Kerscher S, et al. Human ind1, an iron-sulfur cluster assembly factor for respiratory complex I. Mol Cell Biol. 2009;29:6059–6073. MCB.00817-09 [pii];10.1128/MCB.00817-09 [doi] PubMed PMC

Hrdy I, Hirt RP, Dolezal P, Bardonova L, Foster PG, et al. Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I. . Nature. 2004;432:618–622. PubMed

Gelling C, Dawes IW, Richhardt N, Lill R, Mühlenhoff U. Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes. Mol Cell Biol. 2008;28:1851–1861. MCB.01963-07 [pii];10.1128/MCB.01963-07 [doi] PubMed PMC

Mühlenhoff U, Richhardt N, Gerber J, Lill R. Characterization of iron-sulfur protein assembly in isolated mitochondria. A requirement for ATP, NADH, and reduced iron. J Biol Chem. 2002;277:29810–29816. 10.1074/jbc.M204675200 [doi];M204675200 [pii] PubMed

Vernis L, Facca C, Delagoutte E, Soler N, Chanet R, et al. A newly identified essential complex, Dre2-Tah18, controls mitochondria integrity and cell death after oxidative stress in yeast. PLoS One. 2009;4:e4376. 10.1371/journal.pone.0004376 [doi] PubMed PMC

Zhang Y, Lyver ER, Nakamaru-Ogiso E, Yoon H, Amutha B, et al. Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis. Mol Cell Biol. 2008;28:5569–5582. MCB.00642-08 [pii];10.1128/MCB.00642-08 [doi] PubMed PMC

Walsh P, Bursac D, Law YC, Cyr D, Lithgow T. The J-protein family: modulating protein assembly, disassembly and translocation. EMBO Rep. 2004;5:567–571. PubMed PMC

Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N. Importing mitochondrial proteins: machineries and mechanisms. Cell. 2009;138:628–644. S0092-8674(09)00967-2 [pii];10.1016/j.cell.2009.08.005 [doi] PubMed PMC

Horwich AL, Fenton WA, Chapman E, Farr GW. Two families of chaperonin: physiology and mechanism. Annu Rev Cell Dev Biol. 2007;23:115–145. 10.1146/annurev.cellbio.23.090506.123555 [doi] PubMed

Elsner S, Simian D, Iosefson O, Marom M, Azem A. The Mitochondrial Protein Translocation Motor: Structural Conservation between the Human and Yeast Tim14/Pam18-Tim16/Pam16 co-Chaperones. Int J Mol Sci. 2009;10:2041–2053. 10.3390/ijms10052041 [doi] PubMed PMC

Mokranjac D, Bourenkov G, Hell K, Neupert W, Groll M. Structure and function of Tim14 and Tim16, the J and J-like components of the mitochondrial protein import motor. EMBO J. 2006;25:4675–4685. 7601334 [pii];10.1038/sj.emboj.7601334 [doi] PubMed PMC

Schneider A, Bursac D, Lithgow T. The direct route: a simplified pathway for protein import into the mitochondrion of trypanosomes. Trends Cell Biol. 2008;18:12–18. S0962-8924(07)00297-8 [pii];10.1016/j.tcb.2007.09.009 [doi] PubMed

Osborne AR, Rapoport TA, van den Berg B. Protein translocation by the Sec61/SecY channel. Annu Rev Cell Dev Biol. 2005;21:529–550. 10.1146/annurev.cellbio.21.012704.133214 [doi] PubMed

Clements A, Bursac D, Gatsos X, Perry AJ, Civciristov S, et al. The reducible complexity of a mitochondrial molecular machine. Proc Natl Acad Sci U S A. 2009;106:15791–15795. 0908264106 [pii];10.1073/pnas.0908264106 [doi] PubMed PMC

Simpson AG, Inagaki Y, Roger AJ. Comprehensive multigene phylogenies of excavate protists reveal the evolutionary positions of “primitive” eukaryotes. Mol Biol Evol. 2006;23:615–625. msj068 [pii];10.1093/molbev/msj068 [doi] PubMed

van der Laan M, Meinecke M, Dudek J, Hutu DP, Lind M, et al. Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nat Cell Biol. 2007;9:1152–1159. ncb1635 [pii];10.1038/ncb1635 [doi] PubMed

Lev S, Ben HD, Peretti D, Dahan N. The VAP protein family: from cellular functions to motor neuron disease. Trends Cell Biol. 2008;18:282–290. S0962-8924(08)00120-7 [pii];10.1016/j.tcb.2008.03.006 [doi] PubMed

Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, et al. Molecular machinery for non-vesicular trafficking of ceramide. Nature. 2003;426:803–809. 10.1038/nature02188 [doi];nature02188 [pii] PubMed

Reinders J, Zahedi RP, Pfanner N, Meisinger C, Sickmann A. The complete yeast mitochondrial proteome: Multidimensional separation techniques for mitochondrial proteomics. Journal of Proteome Research. 2006;5:1543–1554. PubMed

Waller RF, Jabbour C, Chan NC, Celik N, Likic VA, et al. Evidence of a reduced and modified mitochondrial protein import apparatus in microsporidian mitosomes. Eukaryot Cell. 2009;8:19–26. EC.00313-08 [pii];10.1128/EC.00313-08 [doi] PubMed PMC

Pusnik M, Charriere F, Maser P, Waller RF, Dagley MJ, et al. The single mitochondrial porin of Trypanosoma brucei is the main metabolite transporter in the outer mitochondrial membrane. Mol Biol Evol. 2009;26:671–680. msn288 [pii];10.1093/molbev/msn288 [doi] PubMed

Dolezal P, Dagley MJ, Kono M, Wolynec P, Likic VA, et al. The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica. PLoS Pathog. 2010;6:e1000812. 10.1371/journal.ppat.1000812 [doi] PubMed PMC

Alcock F, Clements A, Webb C, Lithgow T. Evolution. Tinkering inside the organelle. Science. 2010;327:649–650. 327/5966/649 [pii];10.1126/science.1182129 [doi] PubMed

Macasev D, Whelan J, Newbigin E, Silva-Filho MC, Mulhern TD, et al. Tom22′, an 8-kDa trans-site receptor in plants and protozoans, is a conserved feature of the TOM complex that appeared early in the evolution of eukaryotes. Mol Biol Evol. 2004;21:1557–1564. 10.1093/molbev/msh166 [doi];msh166 [pii] PubMed

Kmita H, Budzinska M. Involvement of the TOM complex in external NADH transport into yeast mitochondria depleted of mitochondrial porin1. Biochim Biophys Acta. 2000;1509:86–94. S0005273600002844 [pii] PubMed

Gentle I, Gabriel K, Beech P, Waller R, Lithgow T. The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria. J Cell Biol. 2004;164:19–24. 10.1083/jcb.200310092 [doi];jcb.200310092 [pii] PubMed PMC

Kozjak V, Wiedemann N, Milenkovic D, Lohaus C, Meyer HE, et al. An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane. J Biol Chem. 2003;278:48520–48523. 10.1074/jbc.C300442200 [doi];C300442200 [pii] PubMed

Paschen SA, Waizenegger T, Stan T, Preuss M, Cyrklaff M, et al. Evolutionary conservation of biogenesis of beta-barrel membrane proteins. Nature. 2003;426:862–866. 10.1038/nature02208 [doi];nature02208 [pii] PubMed

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

Gatsos X, Perry AJ, Anwari K, Dolezal P, Wolynec PP, et al. Protein secretion and outer membrane assembly in Alphaproteobacteria. FEMS Microbiol Rev. 2008;32:995–1009. FMR130 [pii];10.1111/j.1574-6976.2008.00130.x [doi] PubMed PMC

Kispal G, Csere P, Guiard B, Lill R. The ABC transporter Atm1p is required for mitochondrial iron homeostasis. Febs Letters. 1997;418:346–350. PubMed

Lange H, Lisowsky T, Gerber J, Mühlenhoff U, Kispal G, et al. An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins. Embo Reports. 2001;2:715–720. PubMed PMC

Chan KW, Slotboom DJ, Cox S, Embley TM, Fabre O, et al. A novel ADP/ATP transporter in the mitosome of the microaerophilic human parasite Entamoeba histolytica. Current Biology. 2005;15:737–742. PubMed

Keister DB. Axenic Culture of Giardia-Lamblia in Tyi-S-33 Medium Supplemented with Bile. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1983;77:487–488. PubMed

Dagley MJ, Dolezal P, Likic VA, Smid O, Purcell AW, et al. The protein import channel in the outer mitosomal membrane of Giardia intestinalis. Mol Biol Evol. 2009;26:1941–1947. msp117 [pii];10.1093/molbev/msp117 [doi] PubMed PMC

Likic VA, Dolezal P, Celik N, Dagley M, Lithgow T. Using hidden markov models to discover new protein transport machines. Methods Mol Biol. 2010;619:271–284. 10.1007/978-1-60327-412-8_16 [doi] PubMed

Sun CH, Chou CF, Tai JH. Stable DNA transfection of the primitive protozoan pathogen Giardia lamblia. Mol Biochem Parasitol. 1998;92:123–132. PubMed

Mlf mediates proteotoxic response via formation of cellular foci for protein folding and degradation in Giardia

Expanded gene and taxon sampling of diplomonads shows multiple switches to parasitic and free-living lifestyle

Comparative analysis of mitochondrion-related organelles in anaerobic amoebozoans

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

The free-living flagellate Paratrimastix pyriformis uses a distinct mitochondrial carrier to balance adenine nucleotide pools

Reduced mitochondria provide an essential function for the cytosolic methionine cycle

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

Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri

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

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

A Uniquely Complex Mitochondrial Proteome from Euglena gracilis

The draft nuclear genome sequence and predicted mitochondrial proteome of Andalucia godoyi, a protist with the most gene-rich and bacteria-like mitochondrial genome

Mitochondrial dynamics in parasitic protists

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

Fe-S cluster assembly in the supergroup Excavata

Giardia intestinalis mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum

Organelles that illuminate the origins of Trichomonas hydrogenosomes and Giardia mitosomes

Conservation of Transit Peptide-Independent Protein Import into the Mitochondrial and Hydrogenosomal Matrix

Probing the Biology of Giardia intestinalis Mitosomes Using In Vivo Enzymatic Tagging