The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus

English version

Autoři: Matthias Misslinger ^aff001; Mareike Thea Scheven ^aff002; Peter Hortschansky ^aff002; Manuel Sánchez López-Berges ^aff001; Katharina Heiss ^aff001; Nicola Beckmann ^aff001; Thomas Heigl ^aff001; Martin Hermann ^aff004; Thomas Krüger ^aff002; Olaf Kniemeyer ^aff002; Axel A. Brakhage ^aff002; Hubertus Haas ^aff001
Působiště autorů: Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria ^aff001; Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology—Hans Knöll Institute (HKI), Jena, Germany ^aff002; Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany ^aff003; Department of Anaesthesiology and Critical Care Medicine, Medical University of Innsbruck, Innsbruck, Austria ^aff004
Vyšlo v časopise: The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus. PLoS Genet 15(9): e32767. doi:10.1371/journal.pgen.1008379
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008379

Souhrn

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus.

Klíčová slova:

Biology and life sciences – Organisms – Eukaryota – Fungi – Fungal molds – Aspergillus – Aspergillus fumigatus – Yeast – Saccharomyces – Saccharomyces cerevisiae – Schizosaccharomyces – Schizosaccharomyces pombe – Microbiology – Medical microbiology – Microbial pathogens – Fungal pathogens – Mycology – Biochemistry – Proteins – DNA-binding proteins – Transcription factors – Regulatory proteins – Amino acids – Sulfur containing amino acids – Cysteine – Protein interactions – Genetics – Gene expression – Gene regulation – Molecular biology – Molecular biology techniques – Molecular biology assays and analysis techniques – Gene expression and vector techniques – Hyperexpression techniques – Medicine and health sciences – Pathology and laboratory medicine – Pathogens – Physical sciences – Chemistry – Chemical compounds – Organic compounds – Carbohydrates – Monosaccharides – Xylose – Organic chemistry – Research and analysis methods – Animal studies – Experimental organism systems – Model organisms – Yeast and fungal models

Zdroje

1. Schrettl M, Kim HS, Eisendle M, Kragl C, Nierman WC, Heinekamp T, et al. SreA-mediated iron regulation in Aspergillus fumigatus. Mol Microbiol 2008;70:27–43. doi: 10.1111/j.1365-2958.2008.06376.x 18721228

2. Schrettl M, Beckmann N, Varga J, Heinekamp T, Jacobsen ID, Jöchl C, et al. HapX-mediated adaption to iron starvation is crucial for virulence of Aspergillus fumigatus. PLoS Pathog 2010;6:e1001124. doi: 10.1371/journal.ppat.1001124 20941352

3. Gsaller F, Hortschansky P, Beattie SR, Klammer V, Tuppatsch K, Lechner BE, et al. The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess. EMBO J 2014;33:2261–76. doi: 10.15252/embj.201489468 25092765

4. Hortschansky P, Haas H, Huber EM, Groll M, Brakhage AA. The CCAAT-binding complex (CBC) in Aspergillus species. Biochim Biophys Acta—Gene Regul Mech 2017;1860:560–70. doi: 10.1016/j.bbagrm.2016.11.008 27939757

5. Schrettl M, Haas H. Iron homeostasis-Achilles’ heel of Aspergillus fumigatus? Curr Opin Microbiol 2011;14:400–5. doi: 10.1016/j.mib.2011.06.002 21724450

6. Outten CE, Albetel A-N. Iron sensing and regulation in Saccharomyces cerevisiae: Ironing out the mechanistic details. Curr Opin Microbiol 2013;16:662–8. doi: 10.1016/j.mib.2013.07.020 23962819

7. Brault A, Mourer T, Labbé S. Molecular basis of the regulation of iron homeostasis in fission and filamentous yeasts. IUBMB Life 2015;67:801–15. doi: 10.1002/iub.1441 26472434

8. Yamaguchi-Iwai Y, Dancis A, Klausner RD. AFT1: a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae. EMBO J 1995;14:1231–9. 7720713

9. Yamaguchi-Iwai Y, Stearman R, Dancis A, Klausner RD. Iron-regulated DNA binding by the AFT1 protein controls the iron regulon in yeast. EMBO J 1996;15:3377–84. 8670839

10. Yun CW, Ferea T, Rashford J, Ardon O, Brown PO, Botstein D, et al. Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake. J Biol Chem 2000;275:10709–15. doi: 10.1074/jbc.275.14.10709 10744769

11. Li L, Bagley D, Ward DM, Kaplan J. Yap5 is an iron-responsive transcriptional activator that regulates vacuolar iron storage in yeast. Mol Cell Biol 2008;28:1326–37. doi: 10.1128/MCB.01219-07 18070921

12. Rietzschel N, Pierik AJ, Bill E, Lill R, Mühlenhoff U. The Basic Leucine Zipper Stress Response Regulator Yap5 Senses High-Iron Conditions by Coordination of [2Fe-2S] Clusters. Mol Cell Biol 2015;35:370–8. doi: 10.1128/MCB.01033-14 25368382

13. Pelletier B, Beaudoin J, Mukai Y, Labbé S. Fep1, an Iron Sensor Regulating Iron Transporter Gene Expression in Schizosaccharomyces pombe. J Biol Chem 2002;277:22950–8. doi: 10.1074/jbc.M202682200 11956219

14. Mercier A, Watt S, Bähler J, Labbé S, Bahler J, Labbe S. Key Function for the CCAAT-Binding Factor Php4 To Regulate Gene Expression in Response to Iron Deficiency in Fission Yeast. Eukaryot Cell 2008;7:493–508. doi: 10.1128/EC.00446-07 18223116

15. Poor CB, Wegner S V, Li H, Dlouhy AC, Schuermann JP, Sanishvili R, et al. Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2. Proc Natl Acad Sci U S A 2014;111:4043–8. doi: 10.1073/pnas.1318869111 24591629

16. Dlouhy AC, Beaudoin J, Labbé S, Outten CE. Schizosaccharomyces pombe Grx4 regulates the transcriptional repressor Php4 via [2Fe–2S] cluster binding. Metallomics 2017;9:1096–105. doi: 10.1039/c7mt00144d 28725905

17. Chi C-B, Tang Y, Zhang J, Dai Y-N, Abdalla M, Chen Y, et al. Structural and biochemical insights into the multiple functions of yeast Grx3. J Mol Biol 2018. doi: 10.1016/j.jmb.2018.02.024 29524511

18. Herrero E, Ros J, Tamarit J, Bellí G. Glutaredoxins in fungi. Photosynth Res 2006;89:127–40. doi: 10.1007/s11120-006-9079-3 16915356

19. Mühlenhoff U, Molik S, Godoy JR, Uzarska M a., Richter N, Seubert A, et al. Cytosolic monothiol glutaredoxins function in intracellular iron sensing and trafficking via their bound iron-sulfur cluster. Cell Metab 2010;12:373–85. doi: 10.1016/j.cmet.2010.08.001 20889129

20. Lillig CH, Berndt C. Glutaredoxins in Thiol/Disulfide Exchange. Antioxid Redox Signal 2013;18:1654–65. doi: 10.1089/ars.2012.5007 23231445

21. Osmani AH, Oakley BR, Osmani SA. Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique. Nat Protoc 2006;1:2517–26. doi: 10.1038/nprot.2006.406 17406500

22. Zadra I, Abt B, Parson W, Haas H. xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE. Appl Environ Microbiol 2000;66:4810–6. doi: 10.1128/aem.66.11.4810-4816.2000 11055928

23. Misslinger M, Lechner BE, Bacher K, Haas H. Iron-sensing is governed by mitochondrial, not by cytosolic iron-sulfur cluster biogenesis in Aspergillus fumigatus. Metallomics 2018;10:1687–700. doi: 10.1039/c8mt00263k 30395137

24. Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H. A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins. Mol Cell Proteomics 2008;7:282–9. doi: 10.1074/mcp.M700342-MCP200 17951627

25. Netz DJA, Pierik AJ, Stümpfig M, Mühlenhoff U, Lill R. The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol. Nat Chem Biol 2007;3:278–86. doi: 10.1038/nchembio872 17401378

26. Netz DJA, Pierik AJ, Stümpfig M, Bill E, Sharma AK, Pallesen LJ, et al. A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation. J Biol Chem 2012;287:12365–78. doi: 10.1074/jbc.M111.328914 22362766

27. Zhang Y, Lyver ER, Nakamaru-Ogiso E, Yoon H, Amutha B, Lee D-WD-W, et al. Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis. Mol Cell Biol 2008;28:5569–82. doi: 10.1128/MCB.00642-08 18625724

28. Netz DJA, Stümpfig M, Doré C, Mühlenhoff U, Pierik AJ, Lill R. Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis. Nat Chem Biol 2010;6:758–65. doi: 10.1038/nchembio.432 20802492

29. Stehling O, Vashisht A a, Mascarenhas J, Jonsson ZO, Sharma T, Netz DJ a, et al. Metabolism and Genomic Integrity 2012;337:195–9. doi: 10.1126/science.1219723.MMS19

30. Balk J, Pierik AJ, Netz DJA, Mühlenhoff U, Lill R. The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins. EMBO J 2004;23:2105–15. doi: 10.1038/sj.emboj.7600216 15103330

31. Jbel M, Mercier A, Labbé S. Grx4 monothiol glutaredoxin is required for iron limitation-dependent inhibition of fep1. Eukaryot Cell 2011;10:629–45. doi: 10.1128/EC.00015-11 21421748

32. Vachon P, Mercier A, Jbel M, Labbé S. The monothiol glutaredoxin Grx4 exerts an iron-dependent inhibitory effect on Php4 function. Eukaryot Cell 2012;11:806–19. doi: 10.1128/EC.00060-12 22523368

33. Wiemann P, Perevitsky A, Lim FY, Shadkchan Y, Knox BP, Landero Figueora JA, et al. Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense. Cell Rep 2017;19:1008–21. doi: 10.1016/j.celrep.2017.04.019 28467895

34. Zhou S, Narukami T, Masuo S, Shimizu M, Fujita T, Doi Y, et al. NO-inducible nitrosothionein mediates NO removal in tandem with thioredoxin. Nat Chem Biol 2013;9:657–63. doi: 10.1038/nchembio.1316 23955366

35. Uzarska MA, Nasta V, Weiler BD, Spantgar F, Ciofi-Baffoni S, Saviello MR, et al. Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins. Elife 2016;5:e16673. doi: 10.7554/eLife.16673 27532772

36. Yu H, Braun P, Yildirim MA, Lemmens I, Venkatesan K, Sahalie J, et al. High-Quality Binary Protein Interaction Map of the Yeast Interactome Network. Science (80-) 2008;322:104–10. doi: 10.1126/science.1158684 18719252

37. Rey P, Taupin-Broggini M, Couturier J, Vignols F, Rouhier N. Is There a Role for Glutaredoxins and BOLAs in the Perception of the Cellular Iron Status in Plants? Front Plant Sci 2019;10:712. doi: 10.3389/fpls.2019.00712 31231405

38. Li H, Mapolelo DT, Randeniya S, Johnson MK, Outten CE. Human glutaredoxin 3 forms [2Fe-2S]-bridged complexes with human BolA2. Biochemistry 2012;51:1687–96. doi: 10.1021/bi2019089 22309771

39. Freibert SA, Weiler BD, Bill E, Pierik AJ, Mühlenhoff U, Lill R. Biochemical Reconstitution and Spectroscopic Analysis of Iron–Sulfur Proteins. Methods Enzymol., vol. 599, 2018, p. 197–226. doi: 10.1016/bs.mie.2017.11.034 29746240

40. Ojeda L, Keller G, Muhlenhoff U, Rutherford JC, Lill R, Winge DR. Role of glutaredoxin-3 and glutaredoxin-4 in the iron regulation of the Aft1 transcriptional activator in Saccharomyces cerevisiae. J Biol Chem 2006;281:17661–9. doi: 10.1074/jbc.M602165200 16648636

41. Kim K-D, Kim H-JJ, Lee K-C, Roe J-H. Multi-domain CGFS-type glutaredoxin Grx4 regulates iron homeostasis via direct interaction with a repressor Fep1 in fission yeast. Biochem Biophys Res Commun 2011;408:609–14. doi: 10.1016/j.bbrc.2011.04.069 21531205

42. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 2009;37:W202–8. doi: 10.1093/nar/gkp335 19458158

43. Mercier A, Labbé S. Both Php4 Function and Subcellular Localization Are Regulated by Iron via a Multistep Mechanism Involving the Glutaredoxin Grx4 and the Exportin Crm1. J Biol Chem 2009;284:20249–62. doi: 10.1074/jbc.M109.009563 19502236

44. Pimentel C, Vicente C, Menezes RA, Caetano S, Carreto L, Rodrigues-Pousada C. The role of the Yap5 transcription factor in remodeling gene expression in response to Fe bioavailability. PLoS One 2012;7:e37434. doi: 10.1371/journal.pone.0037434 22616008

45. Johnson DC, Dean DR, Smith AD, Johnson MK. Structure, Function, and Formation of Biological Iron-Sulfur Clusters. Annu Rev Biochem 2005;74:247–81. doi: 10.1146/annurev.biochem.74.082803.133518 15952888

46. Encinar del Dedo J, Gabrielli N, Carmona M, Ayté J, Hidalgo E. A Cascade of Iron-Containing Proteins Governs the Genetic Iron Starvation Response to Promote Iron Uptake and Inhibit Iron Storage in Fission Yeast. PLOS Genet 2015;11:e1005106. doi: 10.1371/journal.pgen.1005106 25806539

47. Attarian R, Hu G, Sánchez-León E, Caza M, Croll D, Do E, et al. The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence in Cryptococcus neoformans. MBio 2018;9:e02377–18. doi: 10.1128/mBio.02377-18 30514787

48. Pujol-Carrion N, De La Torre-Ruiz MA. Glutaredoxins Grx4 and Grx3 of Saccharomyces cerevisiae play a role in actin dynamics through their trx domains, which contributes to oxidative stress resistance. Appl Environ Microbiol 2010;76:7826–35. doi: 10.1128/AEM.01755-10 20889785

49. Vall-llaura N, Reverter-Branchat G, Vived C, Weertman N, Rodríguez-Colman MJ, Cabiscol E. Reversible glutathionylation of Sir2 by monothiol glutaredoxins Grx3/4 regulates stress resistance. Free Radic Biol Med 2016;96:45–56. doi: 10.1016/j.freeradbiomed.2016.04.008 27085841

50. Inigo S, Nagels Durand A, Ritter A, Le Gall S, Termathe M, Klassen R, et al. Glutaredoxin GRXS17 Associates with the Cytosolic Iron-Sulfur Cluster Assembly Pathway. Plant Physiol 2016;172:858–73. doi: 10.1104/pp.16.00261 27503603

51. Mercier A, Pelletier B, Labbe S. A transcription factor cascade involving Fep1 and the CCAAT-binding factor Php4 regulates gene expression in response to iron deficiency in the fission yeast Schizosaccharomyces pombe. Eukaryot Cell 2006;5:1866–81. doi: 10.1128/EC.00199-06 16963626

52. Jung WH, Sham A, White R, Kronstad JW. Iron Regulation of the Major Virulence Factors in the AIDS-Associated Pathogen Cryptococcus neoformans. PLoS Biol 2006;4:e410. doi: 10.1371/journal.pbio.0040410 17121456

53. Jung WH, Saikia S, Hu G, Wang J, Fung CK, D’Souza C, et al. HapX Positively and Negatively Regulates the Transcriptional Response to Iron Deprivation in Cryptococcus neoformans. PLoS Pathog 2010;6:e1001209. doi: 10.1371/journal.ppat.1001209 21124817

54. Li H, Mapolelo DT, Dingra NN, Keller G, Riggs-Gelasco PJ, Winge DR, et al. Histidine 103 in Fra2 Is an Iron-Sulfur Cluster Ligand in the [2Fe-2S] Fra2-Grx3 Complex and Is Required for in Vivo Iron Signaling in Yeast. J Biol Chem 2011;286:867–76. doi: 10.1074/jbc.M110.184176 20978135

55. Jacques J-F, Mercier A, Brault A, Mourer T, Labbé S. Fra2 Is a Co-Regulator of Fep1 Inhibition in Response to Iron Starvation. PLoS One 2014;9:e98959. doi: 10.1371/journal.pone.0098959 24897379

56. Li H, Outten CE. Monothiol CGFS glutaredoxins and BolA-like proteins: [2Fe-2S] binding partners in iron homeostasis. Biochemistry 2012;51:4377–89. doi: 10.1021/bi300393z 22583368

57. Kim H-J, Lee K-L, Kim K-D, Roe J-H. The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines. Biochem Biophys Res Commun 2016;478:187–92. doi: 10.1016/j.bbrc.2016.07.070 27444384

58. Schrettl M, Bignell E, Kragl C, Joechl C, Rogers T, Arst HN, et al. Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence. J Exp Med 2004;200:1213–9. doi: 10.1084/jem.20041242 15504822

59. Schrettl M, Bignell E, Kragl C, Sabiha Y, Loss O, Eisendle M, et al. Distinct Roles for Intra- and Extracellular Siderophores during Aspergillus fumigatus Infection. PLoS Pathog 2007;3:e128. doi: 10.1371/journal.ppat.0030128 17845073

60. Yasmin S, Alcazar-Fuoli L, Gründlinger M, Puempel T, Cairns T, Blatzer M, et al. Mevalonate governs interdependency of ergosterol and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus. Proc Natl Acad Sci U S A 2012;109:E497–504. doi: 10.1073/pnas.1106399108 22106303

61. McDonagh A, Fedorova ND, Crabtree J, Yu Y, Kim S, Chen D, et al. Sub-Telomere Directed Gene Expression during Initiation of Invasive Aspergillosis. PLoS Pathog 2008;4:e1000154. doi: 10.1371/journal.ppat.1000154 18787699

62. Petzer V, Wermke M, Tymoszuk P, Wolf D, Seifert M, Ovaçin R, et al. Enhanced labile plasma iron in hematopoietic stem cell transplanted patients promotes Aspergillus outgrowth. Blood Adv 2019;3:1695–700. doi: 10.1182/bloodadvances.2019000043 31167821

63. Pontecorvo G, Roper JA, Hemmons LM, Macdonald KD, Bufton AWJ. The genetics of Aspergillus nidulans. Adv Genet 1953;5:141–238. 13040135

64. Bayram O, Krappmann S, Ni M, Bok JW, Helmstaedt K, Valerius O, et al. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 2008;320:1504–6. doi: 10.1126/science.1155888 18556559

65. Mattern IE, Punt PJ, Van den Hondel CAMJJ. A vector for Aspergillus transformation conferring phleomycin resistance. Fungal Genet Rep 1988;35:25. doi: 10.4148/1941-4765.1533

66. Langfelder K, Jahn B, Gehringer H, Schmidt A, Wanner G, Brakhage AA. Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med Microbiol Immunol 1998;187:79–89. 9832321

67. Krappmann S, Jung N, Medic B, Busch S, Prade RA, Braus GH. The Aspergillus nidulans F-box protein GrrA links SCF activity to meiosis. Mol Microbiol 2006;61:76–88. doi: 10.1111/j.1365-2958.2006.05215.x 16824096

68. Nødvig CS, Nielsen JB, Kogle ME, Mortensen UH. A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS One 2015;10:1–18. doi: 10.1371/journal.pone.0133085 26177455

69. Gsaller F, Eisendle M, Lechner BE, Schrettl M, Lindner H, Müller D, et al. The interplay between vacuolar and siderophore-mediated iron storage in Aspergillus fumigatus. Metallomics 2012;4:1262–70. doi: 10.1039/c2mt20179h 23151814

70. Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 1987;56:117–24. doi.org/10.1016/0378-1119(87)90164-8 2824287

71. Gressler M, Zaehle C, Scherlach K, Hertweck C, Brock M. Multifactorial Induction of an Orphan PKS-NRPS Gene Cluster in Aspergillus terreus. Chem Biol 2011;18:198–209. doi: 10.1016/j.chembiol.2010.12.011 21236704

72. Hervás-Aguilar A, Peñalva MA. Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans. Eukaryot Cell 2010;9:1504–18. doi: 10.1128/EC.00119-10 20693304

73. Harlow E, Lane DP. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 1988.

74. López-Berges MS, Pinar M, Abenza JF, Arst HN, Peñalva MA. The Aspergillus nidulans syntaxin PepAPep12 is regulated by two Sec1/Munc-18 proteins to mediate fusion events at early endosomes, late endosomes and vacuoles. Mol Microbiol 2016;99:199–216. doi: 10.1111/mmi.13226 26395371

75. Liu H-L, Osmani AH, Ukil L, Son S, Markossian S, Shen K-F, et al. Single-step affinity purification for fungal proteomics. Eukaryot Cell 2010;9:831–3. doi: 10.1128/EC.00032-10 20363899

76. Diebold M-L, Fribourg S, Koch M, Metzger T, Romier C. Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer. J Struct Biol 2011;175:178–88. doi: 10.1016/j.jsb.2011.02.001 21320604