Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII

Autoři: Mario Pinar aff001;  Ernesto Arias-Palomo aff002;  Vivian de los Ríos aff003;  Herbert N. Arst, Jr aff004;  Miguel A. Peñalva aff001
Působiště autorů: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Madrid, Spain aff001;  Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas CSIC, Madrid, Spain aff002;  Proteomics Facility, Centro de Investigaciones Biológicas CSIC, Madrid, Spain aff003;  Section of Microbiology, Imperial College London, London, United Kingdom aff004
Vyšlo v časopise: Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII. PLoS Genet 15(12): e1008557. doi:10.1371/journal.pgen.1008557
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008557


TRAnsport Protein Particle complexes (TRAPPs) are ubiquitous regulators of membrane traffic mediating nucleotide exchange on the Golgi regulatory GTPases RAB1 and RAB11. In S. cerevisiae and metazoans TRAPPs consist of two large oligomeric complexes: RAB11-activating TRAPPII and RAB1-activating TRAPPIII. These share a common core TRAPPI hetero-heptamer, absent in metazoans but detected in minor proportions in yeast, likely originating from in vitro-destabilized TRAPPII/III. Despite overall TRAPP conservation, the budding yeast genome has undergone extensive loss of genes, and lacks homologues of some metazoan TRAPP subunits. With nearly twice the total number of genes of S. cerevisiae, another ascomycete Aspergillus nidulans has also been used for studies on TRAPPs. We combined size-fractionation chromatography with single-step purification coupled to mass-spectrometry and negative-stain electron microscopy to establish the relative abundance, composition and architecture of Aspergillus TRAPPs, which consist of TRAPPII and TRAPPIII in a 2:1 proportion, plus a minor amount of TRAPPI. We show that Aspergillus TRAPPIII contains homologues of metazoan TRAPPC11, TRAPPC12 and TRAPPC13 subunits, absent in S. cerevisiae, and establish that these subunits are recruited to the complex by Tca17/TRAPPC2L, which itself binds to the ‘Trs33 side’ of the complex. Thus Aspergillus TRAPPs compositionally resemble mammalian TRAPPs to a greater extent than those in budding yeast. Exploiting the ability of constitutively-active (GEF-independent, due to accelerated GDP release) RAB1* and RAB11* alleles to rescue viability of null mutants lacking essential TRAPP subunits, we establish that the only essential role of TRAPPs is activating RAB1 and RAB11, and genetically classify each essential subunit according to their role(s) in TRAPPII (TRAPPII-specific subunits) or TRAPPII and TRAPPIII (core TRAPP subunits). Constitutively-active RAB mutant combinations allowed examination of TRAPP composition in mutants lacking essential subunits, which led to the discovery of a stable Trs120/Trs130/Trs65/Tca17 TRAPPII-specific subcomplex whose Trs20- and Trs33-dependent assembly onto core TRAPP generates TRAPPII.

Klíčová slova:

Aspergillus nidulans – Dimerization – Elution – Protein domains – Saccharomyces cerevisiae – Yeast – Gel filtration – Exocytosis


1. Cai H, Yu S, Menon S, Cai Y, Lazarova D, et al. (2007) TRAPPI tethers COPII vesicles by binding the coat subunit Sec23. Nature 445: 941–944. doi: 10.1038/nature05527 17287728

2. Sacher M, Barrowman J, Wang W, Horecka J, Zhang Y, et al. (2001) TRAPP I Implicated in the Specificity of Tethering in ER-to-Golgi Transport. Mol Cell 7: 433–442. doi: 10.1016/s1097-2765(01)00190-3 11239471

3. Kim YG, Raunser S, Munger C, Wagner J, Song YL, et al. (2006) The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell 127: 817–830. doi: 10.1016/j.cell.2006.09.029 17110339

4. Jones S, Newman C, Liu F, Segev N (2000) The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32. Mol Biol Cell 11: 4403–4411. doi: 10.1091/mbc.11.12.4403 11102533

5. Morozova N, Liang Y, Tokarev AA, Chen SH, Cox R, et al. (2006) TRAPPII subunits are required for the specificity switch of a Ypt-Rab GEF. Nat Cell Biol 8: 1263–1269. doi: 10.1038/ncb1489 17041589

6. Cai Y, Chin HF, Lazarova D, Menon S, Fu C, et al. (2008) The structural basis for activation of the Rab Ypt1p by the TRAPP membrane-tethering complexes. Cell 133: 1202–1213. doi: 10.1016/j.cell.2008.04.049 18585354

7. Pinar M, Arst HN Jr., Pantazopoulou A, Tagua VG, de los Ríos V, et al. (2015) TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 orthologue RabE/RAB11. Proc Natl Acad Sci USA 112: 4346–4351. doi: 10.1073/pnas.1419168112 25831508

8. Thomas LL, Fromme JC (2016) GTPase cross talk regulates TRAPPII activation of Rab11 homologues during vesicle biogenesis. J Cell Biol 215: 499–513. doi: 10.1083/jcb.201608123 27872253

9. Thomas LL, Joiner AMN, Fromme JC (2018) The TRAPPIII complex activates the GTPase Ypt1 (Rab1) in the secretory pathway. J Cell Biol 217: 283–298. doi: 10.1083/jcb.201705214 29109089

10. Thomas LL, van der Vegt SA, Fromme JC (2019) A Steric Gating Mechanism Dictates the Substrate Specificity of a Rab-GEF. Dev Cell 48: 100–114.e109. doi: 10.1016/j.devcel.2018.11.013 30528786

11. Riedel F, Galindo A, Muschalik N, Munro S (2017) The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases. J Cell Biol 217: 601–617. doi: 10.1083/jcb.201705068 29273580

12. Lipatova Z, Segev N (2019) Ypt/Rab GTPases and their TRAPP GEFs at the Golgi. FEBS Letters 593: 2488–2500. doi: 10.1002/1873-3468.13574 31400292

13. Choi C, Davey M, Schluter C, Pandher P, Fang Y, et al. (2011) Organization and assembly of the TRAPPII complex. Traffic 12: 715–725. doi: 10.1111/j.1600-0854.2011.01181.x 21453443

14. Liang Y, Morozova N, Tokarev AA, Mulholland JW, Segev N (2007) The role of Trs65 in the Ypt/Rab guanine nucleotide exchange factor function of the TRAPP II complex. Mol Biol Cell 18: 2533–2541. doi: 10.1091/mbc.E07-03-0221 17475775

15. Lynch-Day MA, Bhandari D, Menon S, Huang J, Cai H, et al. (2010) Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy. Proc Natl Acad Sci USA 107: 7811–7816. doi: 10.1073/pnas.1000063107 20375281

16. Peñalva MA, Galindo A, Abenza JF, Pinar M, Calcagno-Pizarelli AM, et al. (2012) Searching for gold beyond mitosis: mining intracellular membrane traffic in Aspergillus nidulans. Cell Logist 2: 2–14. doi: 10.4161/cl.19304 22645705

17. Peñalva MA, Zhang J, Xiang X, Pantazopoulou A (2017) Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25 and kinesin-1 and is independent of kinesin-3. Mol Biol Cell 28: 947–961. doi: 10.1091/mbc.E16-08-0566 28209731

18. Abenza JF, Pantazopoulou A, Rodríguez JM, Galindo A, Peñalva MA (2009) Long-distance movement of Aspergillus nidulans early endosomes on microtubule tracks. Traffic 10: 57–75. doi: 10.1111/j.1600-0854.2008.00848.x 19000168

19. Abenza JF, Galindo A, Pantazopoulou A, Gil C, de los Ríos V, et al. (2010) Aspergillus RabBRab5 integrates acquisition of degradative identity with the long-distance movement of early endosomes. Mol Biol Cell 21: 2756–2769. doi: 10.1091/mbc.E10-02-0119 20534811

20. Zhang J, Qiu R, Arst HN Jr., Peñalva MA, Xiang X (2014) HookA is a novel dynein-early endosome linker critical for cargo movement in vivo. J Cell Biol 204: 1009–1026. doi: 10.1083/jcb.201308009 24637327

21. Hernández-González M, Bravo-Plaza I, Pinar M, de los Ríos V, Arst HN Jr., et al. (2018) Endocytic recycling via the TGN underlies the polarized hyphal mode of life. PLoS Genetics 14: e1007291. doi: 10.1371/journal.pgen.1007291 29608571

22. Takeshita N, Evangelinos M, Zhou L, Serizawa T, Somera-Fajardo RA, et al. (2017) Pulses of Ca2+ coordinate actin assembly and exocytosis for stepwise cell extension. Proc Natl Acad Sci USA 114: 5701–5706. doi: 10.1073/pnas.1700204114 28507141

23. Pantazopoulou A, Pinar M, Xiang X, Peñalva MA (2014) Maturation of late Golgi cisternae into RabERAB11 exocytic post-Golgi carriers visualized in vivo. Mol Biol Cell 25: 2428–2443. doi: 10.1091/mbc.E14-02-0710 24943841

24. Pinar M, Pantazopoulou A, Arst HN Jr., Peñalva MA (2013) Acute inactivation of the Aspergillus nidulans Golgi membrane fusion machinery: correlation of apical extension arrest and tip swelling with cisternal disorganization. Mol Microbiol 89: 228–248. doi: 10.1111/mmi.12280 23714354

25. Jedd G, Richardson C, Litt R, Segev N (1995) The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway. J Cell Biol 131: 583–590. doi: 10.1083/jcb.131.3.583 7593181

26. Shen XX, Opulente DA, Kominek J, Zhou X, Steenwyk JL, et al. (2018) Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum. Cell 175: 1533–1545 e1520. doi: 10.1016/j.cell.2018.10.023 30415838

27. Sacher M, Shahrzad N, Kamel H, Milev MP (2018) TRAPPopathies, an emerging set of disorders linked to variations in the genes encoding transport protein particle (TRAPP)-associated proteins. Traffic 20: 5–26. doi: 10.1111/tra.12615 30152084

28. Brunet S, Sacher M (2014) Are All Multisubunit Tethering Complexes Bona Fide Tethers? Traffic 15: 1282–1287. doi: 10.1111/tra.12200 25048641

29. Muschalik N, Munro S (2018) Golgins. Curr Biol 28: R374–R376. doi: 10.1016/j.cub.2018.01.006 29689216

30. Yip CK, Berscheminski J, Walz T (2010) Molecular architecture of the TRAPPII complex and implications for vesicle tethering. Nat Struc Mol Biol 17: 1298–1304.

31. Scrivens PJ, Noueihed B, Shahrzad N, Hul S, Brunet S, et al. (2011) C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking. Mol Biol Cell 22: 2083–2093. doi: 10.1091/mbc.E10-11-0873 21525244

32. Matalonga L, Bravo M, Serra-Peinado C, García-Pelegrí E, Ugarteburu O, et al. (2017) Mutations in TRAPPC11 are associated with a congenital disorder of glycosylation. Human Mutation 38: 148–151. doi: 10.1002/humu.23145 27862579

33. Ramírez-Peinado S, Ignashkova TI, van Raam BJ, Baumann J, Sennott EL, et al. (2017) TRAPPC13 modulates autophagy and the response to Golgi stress. J Cell Sci 130: 2251–2265. doi: 10.1242/jcs.199521 28536105

34. Stanga D, Zhao Q, Milev MP, Saint-Dic D, Jimenez-Mallebrera C, et al. (2019) TRAPPC11 functions in autophagy by recruiting ATG2B-WIPI4/WDR45 to preautophagosomal membranes. Traffic 20: 325–345. doi: 10.1111/tra.12640 30843302

35. Taussig D, Lipatova Z, Kim JJ, Zhang X, Segev N (2013) Trs20 is Required for TRAPP II Assembly. Traffic 14: 678–690. doi: 10.1111/tra.12065 23465091

36. Zong M, Wu XG, Chan CW, Choi MY, Chan HC, et al. (2011) The adaptor function of TRAPPC2 in mammalian TRAPPs explains TRAPPC2-associated SEDT and TRAPPC9-associated congenital intellectual disability. PLoS One 6: e23350. doi: 10.1371/journal.pone.0023350 21858081

37. Tan D, Cai Y, Wang J, Zhang J, Menon S, et al. (2013) The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway. Proc Natl Acad Sci USA 110: 19432–19437. doi: 10.1073/pnas.1316356110 24218626

38. Lipatova Z, Belogortseva N, Zhang XQ, Kim J, Taussig D, et al. (2012) Regulation of selective autophagy onset by a Ypt/Rab GTPase module. Proc Natl Acad Sci USA 109: 6981–6986. doi: 10.1073/pnas.1121299109 22509044

39. Taussig D, Lipatova Z, Segev N (2014) Trs20 is required for TRAPP III complex assembly at the PAS and its function in autophagy. Traffic 15: 327–337. doi: 10.1111/tra.12145 24329977

40. Pinar M, Pantazopoulou A, Peñalva MA (2013) Live-cell imaging of Aspergillus nidulans autophagy: RAB1 dependence, Golgi independence and ER involvement. Autophagy 9: 1024–1043. doi: 10.4161/auto.24483 23722157

41. Tokarev AA, Taussig D, Sundaram G, Lipatova Z, Liang Y, et al. (2009) TRAPP II complex assembly requires Trs33 or Trs65. Traffic 10: 1831–1844. doi: 10.1111/j.1600-0854.2009.00988.x 19843283

42. Montpetit B, Conibear E (2009) Identification of the Novel TRAPP Associated Protein Tca17. Traffic 10: 713–723. doi: 10.1111/j.1600-0854.2009.00895.x 19220810

43. Wang C, Gohlke U, Roske Y, Heinemann U (2014) Crystal structure of the yeast TRAPP-associated protein Tca17. FEBS J 281: 4195–4206. doi: 10.1111/febs.12888 24961828

44. Scrivens PJ, Shahrzad N, Moores A, Morin A, Brunet S, et al. (2009) TRAPPC2L is a novel, highly conserved TRAPP-interacting protein. Traffic 10: 724–736. doi: 10.1111/j.1600-0854.2009.00906.x 19416478

45. Milev MP, Graziano C, Karall D, Kuper WFE, Al-Deri N, et al. (2018) Bi-allelic mutations in TRAPPC2L result in a neurodevelopmental disorder and have an impact on RAB11 in fibroblasts. Journal of Medical Genetics.

46. Lipatova Z, Majumdar U, Segev N (2016) Trs33-Containing TRAPP IV: A Novel Autophagy-Specific Ypt1 GEF. Genetics 204: 1117–1128. doi: 10.1534/genetics.116.194910 27672095

47. Lord C, Bhandari D, Menon S, Ghassemian M, Nycz D, et al. (2011) Sequential interactions with Sec23 control the direction of vesicle traffic. Nature 473: 181–186. doi: 10.1038/nature09969 21532587

48. Zou S, Liu Y, Zhang XQ, Chen Y, Ye M, et al. (2012) Modular TRAPP complexes regulate intracellular protein trafficking through multiple Ypt/Rab GTPases in Saccharomyces cerevisiae. Genetics 191: 451–460. doi: 10.1534/genetics.112.139378 22426882

49. Pinar M, Peñalva MA (2017) Aspergillus nidulans BapH is a RAB11 effector that connects membranes in the Spitzenkörper with basal autophagy. Mol Microbiol 106: 452–468. doi: 10.1111/mmi.13777 28857357

50. Hernández-González M, Pantazopoulou A, Spanoudakis D, Seegers CLC, Peñalva MA (2018) Genetic dissection of the secretory route followed by a fungal extracellular glycosyl hydrolase. Mol Microbiol 109: 781–800. doi: 10.1111/mmi.14073 29995994

51. Todd RB, Davis MA, Hynes MJ (2007) Genetic manipulation of Aspergillus nidulans: meiotic progeny for genetic analysis and strain construction. Nat Protoc 2: 811–821. doi: 10.1038/nprot.2007.112 17446881

52. Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, et al. (1983) Transformation by integration in Aspergillus nidulans. Gene 26: 205–211. doi: 10.1016/0378-1119(83)90191-9 6368319

53. Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, et al. (2006) Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc 1: 3111–3120. doi: 10.1038/nprot.2006.405 17406574

54. Liu HL, Osmani AH, Ukil L, Son S, Markossian S, et al. (2010) Single-step affinity purification for fungal proteomics. Eukaryot Cell 9: 831–833. doi: 10.1128/EC.00032-10 20363899

55. Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, et al. (2005) A versatile and efficient gene targeting system for Aspergillus nidulans. Genetics 172: 1557–1566. doi: 10.1534/genetics.105.052563 16387870

56. Hernández-González M, Bravo-Plaza I, de Los Ríos V, Pinar M, Pantazopoulou A, et al. (2019) COPI localizes to the early Golgi in Aspergillus nidulans. Fungal Genet Biol 123: 78–86. doi: 10.1016/j.fgb.2018.12.003 30550852

57. Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ (2007) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4: 923–925. doi: 10.1038/nmeth1113 17952086

58. Simon I, Zerial M, Goody RS (1996) Kinetics of interaction of Rab5 and Rab7 with nucleotides and magnesium ions. J Biol Chem 271: 20470–20478. doi: 10.1074/jbc.271.34.20470 8702787

59. Osmani AH, Oakley BR, Osmani SA (2006) Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique. Nat Protoc 1: 2517–2526. doi: 10.1038/nprot.2006.406 17406500

60. Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, et al. (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157: 38–46. doi: 10.1016/j.jsb.2006.05.009 16859925

61. Scheres SH (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180: 519–530. doi: 10.1016/j.jsb.2012.09.006 23000701

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