A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

English version České info

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Vyšlo v časopise: A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor. PLoS Pathog 7(12): e32767. doi:10.1371/journal.ppat.1002392
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002392

Souhrn

Zdroje

1. RalphSAvan DoorenGGWallerRFCrawfordMJFraunholzMJ 2004 Tropical infectious diseases: metabolic maps and functions of the Plasmodium falciparum apicoplast. Nat Rev Microbiol 2 203 216

2. SeeberFSoldati-FavreD 2010 Metabolic pathways in the apicoplast of apicomplexa. Int Rev Cell Mol Biol 281 161 228

3. MazumdarJEHWMasekKCAHStriepenB 2006 Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. Proc Natl Acad Sci U S A 103 13192 13197

4. VaughanAMO'NeillMTTarunASCamargoNPhuongTM 2009 Type II fatty acid synthesis is essential only for malaria parasite late liver stage development. Cell Microbiol 11 506 520

5. YuMKumarTRNkrumahLJCoppiARetzlaffS 2008 The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites. Cell Host Microbe 4 567 578

6. BrooksCFJohnsenHvan DoorenGGMuthalagiMLinSS 2010 The toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival. Cell Host Microbe 7 62 73

7. JomaaHWiesnerJSanderbrandSAltincicekBWeidemeyerC 1999 Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285 1573 1576

8. YehEDeRisiJL 2011 Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage P. falciparum. PLoS Biol 9 e1001138

9. NairSCBrooksCFGoodmanCDStrurmAMcFaddenGI 2011 Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii. J Exp Med 208 1547 1559

10. ObornikMJanouskovecJChrudimskyTLukesJ 2009 Evolution of the apicoplast and its hosts: from heterotrophy to autotrophy and back again. Int J Parasitol 39 1 12

11. Cavalier-SmithT 1999 Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the eukaryote family tree. J Eukaryot Microbiol 46 347 366

12. GrayMW 2010 Rethinking plastid evolution. EMBO Rep 11 562 563

13. AgrawalSvan DoorenGGBeattyWLStriepenB 2009 Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins. J Biol Chem 284 33683 33691

14. FastNMKissingerJCRoosDSKeelingPJ 2001 Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids. Mol Biol Evol 18 418 426

15. HarperJTKeelingPJ 2003 Nucleus-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids. Mol Biol Evol 20 1730 1735

16. PatronNJRogersMBKeelingPJ 2004 Gene replacement of fructose-1,6-bisphosphate aldolase supports the hypothesis of a single photosynthetic ancestor of chromalveolates. Eukaryot Cell 3 1169 1175

17. ReumannSDavila-AponteJKeegstraK 1999 The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog. Proc Natl Acad Sci U S A 96 784 789

18. DelwicheCF 1999 Tracing the Thread of Plastid Diversity through the Tapestry of Life. Am Nat 154 S164 S177

19. GouldSBWallerRFMcFaddenGI 2008 Plastid evolution. Annu Rev Plant Biol 59 491 517

20. van DoorenGGSchwartzbachSDOsafuneTMcFaddenGI 2001 Translocation of proteins across the multiple membranes of complex plastids. Biochim Biophys Acta 1541 34 53

21. WallerRFKeelingPJDonaldRGStriepenBHandmanE 1998 Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc Natl Acad Sci U S A 95 12352 12357

22. WallerRFReedMBCowmanAFMcFaddenGI 2000 Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway. EMBO J 19 1794 1802

23. DeRocherAECoppensIKarnatakiAGilbertLARomeME 2008 A thioredoxin family protein of the apicoplast periphery identifies abundant candidate transport vesicles in Toxoplasma gondii. Eukaryot Cell 7 1518 1529

24. KarnatakiADerocherACoppensINashCFeaginJE 2007 Cell cycle-regulated vesicular trafficking of Toxoplasma APT1, a protein localized to multiple apicoplast membranes. Mol Microbiol 63 1653 1668

25. KarnatakiADerocherAECoppensIFeaginJEParsonsM 2007 A membrane protease is targeted to the relict plastid of toxoplasma via an internal signal sequence. Traffic 8 1543 1553

26. LimLKalanonMMcFaddenGI 2009 New proteins in the apicoplast membranes: time to rethink apicoplast protein targeting. Trends Parasitol 25 197 200

27. TawkLDubremetzJFMontcourrierPChicanneGMerezegueF 2011 Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis. PLoS Pathog 7 e1001286

28. KalanonMTonkinCJMcFaddenGI 2009 Characterization of two putative protein translocation components in the apicoplast of Plasmodium falciparum. Eukaryot Cell 8 1146 1154

29. SommerMSGouldSBLehmannPGruberAPrzyborskiJM 2007 Der1-mediated preprotein import into the periplastid compartment of chromalveolates? Mol Biol Evol 24 918 928

30. SporkSHissJAMandelKSommerMKooijTW 2009 An unusual ERAD-like complex is targeted to the apicoplast of Plasmodium falciparum. Eukaryot Cell 8 1134 1145

31. BullmannLHaarmannRMirusOBredemeierRHempelF 2010 Filling the gap, evolutionarily conserved Omp85 in plastids of chromalveolates. J Biol Chem 285 6848 6856

32. van DoorenGGTomovaCAgrawalSHumbelBMStriepenB 2008 Toxoplasma gondii Tic20 is essential for apicoplast protein import. Proc Natl Acad Sci U S A 105 13574 13579

33. FothBJRalphSATonkinCJStruckNSFraunholzM 2003 Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science 299 705 708

34. BozdechZLlinasMPulliamBLWongEDZhuJ 2003 The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 1 E5

35. Anderson-WhiteBRIveyFDChengKSzatanekTLorestaniA 2011 A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii. Cell Microbiol 13 18 31

36. BehnkeMSWoottonJCLehmannMMRadkeJBLucasO 2010 Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5 e12354

37. FoxBARistucciaJGGigleyJPBzikDJ 2009 Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining. Eukaryot Cell 8 520 529

38. HuynhMHCarruthersVB 2009 Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot Cell 8 530 539

39. DarMASharmaAMondalNDharSK 2007 Molecular cloning of apicoplast-targeted Plasmodium falciparum DNA gyrase genes: unique intrinsic ATPase activity and ATP-independent dimerization of PfGyrB subunit. Eukaryot Cell 6 398 412

40. Raghu RamEVKumarABiswasSChaubeySSiddiqiMI 2007 Nuclear gyrB encodes a functional subunit of the Plasmodium falciparum gyrase that is involved in apicoplast DNA replication. Mol Biochem Parasitol 154 30 39

41. BriggsGSMahdiAAWellerGRWenQLloydRG 2004 Interplay between DNA replication, recombination and repair based on the structure of RecG helicase. Philos Trans R Soc Lond B Biol Sci 359 49 59

42. SantosJMFreirePVicenteMArraianoCM 1999 The stationary-phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Mol Microbiol 32 789 798

43. YeHAbdel-GhanySEAndersonTDPilon-SmitsEAPilonM 2006 CpSufE activates the cysteine desulfurase CpNifS for chloroplastic Fe-S cluster formation. J Biol Chem 281 8958 8969

44. ArchibaldJM 2007 Nucleomorph genomes: structure, function, origin and evolution. Bioessays 29 392 402

45. QuanSSchneiderIPanJVon HachtABardwellJC 2007 The CXXC motif is more than a redox rheostat. J Biol Chem 282 28823 28833

46. KilianOKrothPG 2005 Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids. Plant J 41 175 183

47. Nicole PoulsenPMCNilsKroger 2006 Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). J Phycol 42 1059 1065

48. MoogDStorkSZaunerSMaierUG 2011 In silico and in vivo investigations of proteins of a minimized eukaryotic cytoplasm. Genome Biol Evol 3 375 382

49. Herm-GotzAAgop-NersesianCMunterSGrimleyJSWandlessTJ 2007 Rapid control of protein level in the apicomplexan Toxoplasma gondii. Nat Methods 4 1003 1005

50. MeissnerMBrechtSBujardHSoldatiD 2001 Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii. Nucleic Acids Res 29 E115

51. MeissnerMSchluterDSoldatiD 2002 Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298 837 840

52. RadkeJRGueriniMNWhiteMW 2000 Toxoplasma gondii: characterization of temperature-sensitive tachyzoite cell cycle mutants. Exp Parasitol 96 168 177

53. van PoppelNFWelagenJDuistersRFVermeulenANSchaapD 2006 Tight control of transcription in Toxoplasma gondii using an alternative tet repressor. Int J Parasitol 36 443 452

54. GubbelsMJLiCStriepenB 2003 High-throughput growth assay for Toxoplasma gondii using yellow fluorescent protein. Antimicrob Agents Chemother 47 309 316

55. van DoorenGGReiffSBTomovaCMeissnerMHumbelBM 2009 A novel dynamin-related protein has been recruited for apicoplast fission in Toxoplasma gondii. Curr Biol 19 267 276

56. Thomsen-ZiegerNSchachtnerJSeeberF 2003 Apicomplexan parasites contain a single lipoic acid synthase located in the plastid. FEBS Lett 547 80 86

57. GouldSBSommerMSKrothPGGileGHKeelingPJ 2006 Nucleus-to-nucleus gene transfer and protein retargeting into a remnant cytoplasm of cryptophytes and diatoms. Mol Biol Evol 23 2413 2422

58. PatronNJWallerRFArchibaldJMKeelingPJ 2005 Complex protein targeting to dinoflagellate plastids. J Mol Biol 348 1015 1024

59. PatronNJWallerRFKeelingPJ 2006 A tertiary plastid uses genes from two endosymbionts. J Mol Biol 357 1373 1382

60. NishiMHuKMurrayJMRoosDS 2008 Organellar dynamics during the cell cycle of Toxoplasma gondii. J Cell Sci 121 1559 1568

61. DouglasSZaunerSFraunholzMBeatonMPennyS 2001 The highly reduced genome of an enslaved algal nucleus. Nature 410 1091 1096

62. AgrawalSStriepenB 2011 More membranes, more proteins: complex protein import mechanisms into secondary plastids. Protist 161 672 687

63. DangoorIPeled-ZehaviHLevitanAPasandODanonA 2009 A small family of chloroplast atypical thioredoxins. Plant Physiol 149 1240 1250

64. BalseraMSollJBuchananBB 2010 Redox extends its regulatory reach to chloroplast protein import. Trends Plant Sci 15 515 521

65. BeckerTHritzJVogelMCaliebeABukauB 2004 Toc12, a novel subunit of the intermembrane space preprotein translocon of chloroplasts. Mol Biol Cell 15 5130 5144

66. StengelABenzJPBuchananBBSollJBolterB 2009 Preprotein import into chloroplasts via the Toc and Tic complexes is regulated by redox signals in Pisum sativum. Mol Plant 2 1181 1197

67. Striepen BorisSD 2007 enetic manipulation of Toxoplasma gondii. Toxoplasma gondii: The model Apicomplexan Perspective and methods Oxford Elsevier 391 418

68. SoldatiDKimKKampmeierJDubremetzJFBoothroydJC 1995 Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection. Mol Biochem Parasitol 74 87 97

69. GissotMKellyKAAjiokaJWGreallyJMKimK 2007 Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii. PLoS Pathog 3 e77

70. SheinerLDowseTJSoldati-FavreD 2008 Identification of trafficking determinants for polytopic rhomboid proteases in Toxoplasma gondii. Traffic 9 665 677

71. BastinPBagherzadehZMatthewsKRGullK 1996 A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei. Mol Biochem Parasitol 77 235 239

72. PinoPAebyEFothBJSheinerLSoldatiT 2010 Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation in Apicomplexa. Mol Microbiol 76 706 718

73. GubbelsMJWiefferMStriepenB 2004 Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among Apicomplexa. Mol Biochem Parasitol 137 99 110

74. BrossierFStarnesGLBeattyWLSibleyLD 2008 Microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma gondii. Eukaryot Cell 7 664 674

75. Jerka-DziadoszMJenkinsLMNelsenEMWilliamsNEJaeckel-WilliamsR 1995 Cellular polarity in ciliates: persistence of global polarity in a disorganized mutant of Tetrahymena thermophila that disrupts cytoskeletal organization. Dev Biol 169 644 661