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The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain

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Abstract

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8–10 aa)C signature motif. Structure–function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.

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Abbreviations

CAT:

Chloramphenicol acetyl transferase

ECL:

Extra-cellular loop

ER:

Endoplasmic reticulum

GFP:

Green fluorescent protein

MFD:

Membrane fusion domain

PDZ:

Post-synaptic density protein PSD95, Drosophila disc large tumour suppressor Dlg1, and zonula occludens-1 zo-1 proteins

TJ:

Tight junction

TMD:

Trans-membrane Domain

WT:

Wild-type

References

  1. Chen EH, Grote E, Mohler W, Vignery A (2007) Cell-cell fusion. FEBS Lett 581(11):2181–2193

    Article  CAS  PubMed  Google Scholar 

  2. Aguilar PS, Baylies MK, Fleissner A, Helming L, Inoue N, Podbilewicz B, Wang H, Wong M (2013) Genetic basis of cell-cell fusion mechanisms. Trends Genet 29(7):427–437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ydenberg CA, Rose MD (2008) Yeast mating: a model system for studying cell and nuclear fusion. Methods Mol Biol (Clifton, NJ) 475:3–20

    Article  Google Scholar 

  4. Merlini L, Dudin O, Martin SG (2013) Mate and fuse: how yeast cells do it. Open Biol 3(3):130008

    Article  PubMed  PubMed Central  Google Scholar 

  5. Martin SG (2016) Role and organization of the actin cytoskeleton during cell-cell fusion. Semin Cell Dev Biol 60:121–126

    Article  CAS  PubMed  Google Scholar 

  6. White JM, Rose MD (2001) Yeast mating: getting close to membrane merger. Curr Biol 11(1):R16–R20

    Article  CAS  PubMed  Google Scholar 

  7. Heiman MG, Walter P (2000) Prm1p, a pheromone-regulated multispanning membrane protein, facilitates plasma membrane fusion during yeast mating. J Cell Biol 151:719–730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Fleissner A, Diamond S, Glass NL (2009) The Saccharomyces cerevisiae PRM1 homolog in Neurospora crassa is involved in vegetative and sexual cell fusion events but also has postfertilization functions. Genetics 181(2):497–510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Curto MA, Sharifmoghadam MR, Calpena E, De Leon N, Hoya M, Doncel C, Leatherwood J, Valdivieso MH (2014) Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1. Genetics 196(4):1059–1076

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Clemente-Ramos JA, Martin-Garcia R, Sharifmoghadam MR, Konomi M, Osumi M, Valdivieso MH (2009) The tetraspan protein Dni1p is required for correct membrane organization and cell wall remodelling during mating in Schizosaccharomyces pombe. Mol Microbiol 73(4):695–709

    Article  CAS  PubMed  Google Scholar 

  11. Muller EM, Mackin NA, Erdman SE, Cunningham KW (2003) Fig 1p facilitates Ca2+ influx and cell fusion during mating of Saccharomyces cerevisiae. J Biol Chem 278:38461–38469

    Article  CAS  PubMed  Google Scholar 

  12. Proszynski TJ, Klemm R, Bagnat M, Gaus K, Simons K (2006) Plasma membrane polarization during mating in yeast cells. J Cell Biol 173(6):861–866

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bagnat M, Simons K (2002) Cell surface polarization during yeast mating. Proc Natl Acad Sci USA 99(22):14183–14188

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Olmo VN, Grote E (2010) Prm1 targeting to contact sites enhances fusion during mating in Saccharomyces cerevisiae. Eukaryot Cell 9(10):1538–1548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Anderson RG, Jacobson K (2002) A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296(5574):1821–1825

    Article  CAS  PubMed  Google Scholar 

  16. Laude AJ, Prior IA (2004) Plasma membrane microdomains: organization, function and trafficking. Mol Membr Biol 21(3):193–205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Garcia-Parajo MF, Cambi A, Torreno-Pina JA, Thompson N, Jacobson K (2014) Nanoclustering as a dominant feature of plasma membrane organization. J Cell Sci 127(Pt 23):4995–5005

    Article  PubMed  PubMed Central  Google Scholar 

  18. Corcoran JA, Salsman J, de Antueno R, Touhami A, Jericho MH, Clancy EK, Duncan R (2006) The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains. J Biol Chem 281(42):31778–31789

    Article  CAS  PubMed  Google Scholar 

  19. Olivera-Couto A, Aguilar PS (2012) Eisosomes and plasma membrane organization. Mol Genet Genom 287(8):607–620

    Article  CAS  Google Scholar 

  20. Roth MG (2004) Phosphoinositides in constitutive membrane traffic. Physiol Rev 84(3):699–730

    Article  CAS  PubMed  Google Scholar 

  21. Rubinstein E (2011) The complexity of tetraspanins. Biochem Soc Trans 39(2):501–505

    Article  CAS  PubMed  Google Scholar 

  22. Trimble WS, Grinstein S (2015) Barriers to the free diffusion of proteins and lipids in the plasma membrane. J Cell Biol 208(3):259–271

    Article  PubMed  PubMed Central  Google Scholar 

  23. Valdez-Taubas J, Pelham HR (2003) Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling. Curr Biol 13(18):1636–1640

    Article  CAS  PubMed  Google Scholar 

  24. Balda MS, Matter K (2016) Tight junctions as regulators of tissue remodelling. Curr Opin Cell Biol 42:94–101

    Article  CAS  PubMed  Google Scholar 

  25. Lahiri S, Toulmay A, Prinz WA (2015) Membrane contact sites, gateways for lipid homeostasis. Curr Opin Cell Biol 33:82–87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Simons K, Fuller SD (1985) Cell surface polarity in epithelia. Annu Rev Cell Biol 1:243–288

    Article  CAS  PubMed  Google Scholar 

  27. Gunzel D, Yu AS (2013) Claudins and the modulation of tight junction permeability. Physiol Rev 93(2):525–569

    Article  PubMed  PubMed Central  Google Scholar 

  28. Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B, Hatziioannou T, McKeating JA, Bieniasz PD, Rice CM (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446(7137):801–805

    Article  CAS  PubMed  Google Scholar 

  29. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141(7):1539–1550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, Blasig IE (2008) Structure and function of claudins. Biochem Biophys Acta 1778(3):631–645

    Article  CAS  PubMed  Google Scholar 

  31. Krause G, Protze J, Piontek J (2015) Assembly and function of claudins: structure-function relationships based on homology models and crystal structures. Semin Cell Dev Biol 42:3–12

    Article  CAS  PubMed  Google Scholar 

  32. Lingaraju A, Long TM, Wang Y, Austin JR 2nd, Turner JR (2015) Conceptual barriers to understanding physical barriers. Semin Cell Dev Biol 42:13–21

    Article  PubMed  PubMed Central  Google Scholar 

  33. Aguilar PS, Engel A, Walter P (2007) The plasma membrane proteins Prm1 and Fig 1 ascertain fidelity of membrane fusion during yeast mating. Mol Biol Cell 18(2):547–556

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Alvarez FJ, Douglas LM, Rosebrock A, Konopka JB (2008) The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans. Mol Biol Cell 19(12):5214–5225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Forsburg SL, Rhind N (2006) Basic methods for fission yeast. Yeast 23(3):173–183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194:795–823

    Article  CAS  PubMed  Google Scholar 

  37. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. CSHL Press, New York

    Google Scholar 

  38. Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382

    Article  CAS  PubMed  Google Scholar 

  39. Maundrell K (1993) Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123:127–130

    Article  CAS  PubMed  Google Scholar 

  40. Stern B, Nurse P (1997) Fission yeast pheromone blocks S-phase by inhibiting the G1 cyclinB-p34cdc2 kinase. EMBO J 16:534

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Sharifmoghadam MR, Valdivieso MH (2009) The fission yeast SEL1 domain protein Cfh3p: a novel regulator of the glucan synthase Bgs1p whose function is more relevant under stress conditions. J Biol Chem 284(17):11070–11079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. de Leon N, Hoya M, Curto MA, Moro S, Yanguas F, Doncel C, Valdivieso MH (2016) The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast. Mol Microbiol 100(3):409–424

    Article  PubMed  Google Scholar 

  43. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Russ WP, Engelman DM (1999) TOXCAT: a measure of transmembrane helix association in a biological membrane. Proc Natl Acad Sci USA 96(3):863–868

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Armstrong CR (1858) Senes A (2016) Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay. Biochem Biophys Acta 11:2573–2583

    Google Scholar 

  46. Dudin O, Bendezu FO, Groux R, Laroche T, Seitz A, Martin SG (2015) A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast. J Cell Biol 208(7):897–911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lock A, Forfar R, Weston C, Bowsher L, Upton GJ, Reynolds CA, Ladds G, Dixon AM (2014) One motif to bind them: a small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs. Biochem Biophys Acta 1838(12):3036–3051

    Article  CAS  PubMed  Google Scholar 

  48. Hsin J, LaPointe LM, Kazy A, Chipot C, Senes A, Schulten K (2011) Oligomerization state of photosynthetic core complexes is correlated with the dimerization affinity of a transmembrane helix. J Am Chem Soc 133(35):14071–14081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Senes A, Gerstein M, Engelman DM (2000) Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions. J Mol Biol 296(3):921–936

    Article  CAS  PubMed  Google Scholar 

  50. Gong Y, Renigunta V, Zhou Y, Sunq A, Wang J, Yang J, Renigunta A, Baker LA, Hou J (2015) Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization. Mol Biol Cell 26(24):4333–4346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rossa J, Ploeger C, Vorreiter F, Saleh T, Protze J, Gunzel D, Wolburg H, Krause G, Piontek J (2014) Claudin-3 and claudin-5 protein folding and assembly into the tight junction are controlled by non-conserved residues in the transmembrane 3 (TM3) and extracellular loop 2 (ECL2) segments. J Biol Chem 289(11):7641–7653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Teese MG, Langosch D (2015) Role of GxxxG motifs in transmembrane domain interactions. Biochemistry 54(33):5125–5135

    Article  CAS  PubMed  Google Scholar 

  53. Koval M (2013) Differential pathways of claudin oligomerization and integration into tight junctions. Tissue Barriers 1(3):e24518

    Article  PubMed  PubMed Central  Google Scholar 

  54. Iwaki T, Tanaka N, Takagi H, Giga-Hama Y, Takegawa K (2004) Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe. Yeast 21(10):867–881

    Article  CAS  PubMed  Google Scholar 

  55. Wachtler V, Rajagopalan S, Balasubramanian MK (2003) Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe. J Cell Sci 116(Pt 5):867–874

    Article  CAS  PubMed  Google Scholar 

  56. Krug SM, Schulzke JD, Fromm M (2014) Tight junction, selective permeability, and related diseases. Semin Cell Dev Biol 36:166–176

    Article  CAS  PubMed  Google Scholar 

  57. Simske JS (2013) Claudins reign: the claudin/EMP/PMP22/gamma channel protein family in C. elegans. Tissue Barriers 1(3):25502

    Article  Google Scholar 

  58. Hou J, Renigunta A, Gomes AS, Hou M, Paul DL, Waldegger S, Goodenough DA (2009) Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium. Proc Natl Acad Sci USA 106(36):15350–15355

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Zihni C, Mills C, Matter K, Balda MS (2016) Tight junctions: from simple barriers to multifunctional molecular gates. Nat Rev 17(9):564–580

    Article  CAS  Google Scholar 

  60. Lambert D, O’Neill CA, Padfield PJ (2007) Methyl-beta-cyclodextrin increases permeability of Caco-2 cell monolayers by displacing specific claudins from cholesterol rich domains associated with tight junctions. Cell Physiol Biochem 20(5):495–506

    Article  CAS  PubMed  Google Scholar 

  61. Krause SA, Xu H, Gray JV (2008) The synthetic genetic network around PKC1 identifies novel modulators and components of protein kinase C signaling in Saccharomyces cerevisiae. Eukaryot Cell 7(11):1880–1887

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Markvoort AJ, Marrink SJ (2011) Lipid acrobatics in the membrane fusion arena. Curr Top Membr 68:259–294

    Article  CAS  PubMed  Google Scholar 

  63. Lee DB, Jamgotchian N, Allen SG, Abeles MB, Ward HJ (2008) A lipid-protein hybrid model for tight junction. Am J Physiol Renal Physiol 295(6):F1601–F1612

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Nguyen VS, Jobichen C, Tan KW, Tan YW, Chan SL, Ramesh K, Yuan Y, Hong Y, Seetharaman J, Leung KY, Sivaraman J, Mok YK (2015) Structure of AcrH-AopB chaperone-translocator complex reveals a role for membrane hairpins in type III secretion system translocon assembly. Structure 23(11):2022–2031

    Article  CAS  PubMed  Google Scholar 

  65. Hemler ME (2008) Targeting of tetraspanin proteins–potential benefits and strategies. Nat Rev Drug Discov 7(9):747–758

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Sourisseau M, Michta ML, Zony C, Israelow B, Hopcraft SE, Narbus CM, Parra Martin A, Evans MJ (2013) Temporal analysis of hepatitis C virus cell entry with occludin directed blocking antibodies. PLoS Pathog 9(3):e1003244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank E. Keck for the English revision. We are indebted to S. Moreno, D. Mulvihill, O. Nielsen, P. Nurse, P. Pérez, J.C. Ribas, Y. Sanchez, C. Shimoda, and K. Takegawa for strains and plasmids. A. Senes and C. Armstrong are acknowledged for providing plasmids and the guidance to perform the TOXCAT analyses. Financial support from the Ministerio de Economía y Competitividad (MINECO, Spain)/European Union FEDER program (BFU2013-48582-C2-2-P) and Junta de Castilla y León (Grant SA073U14) made this work possible. MAC, SM, and FY were supported by FPU fellowships from the Ministerio de Educación. CGG was supported by the Sistema de Garantía Juvenil program from the Ministerio de Empleo y Seguridad Social, Spain.

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  1. Sandra Moro and Francisco Yanguas have contributed equally to this work.

Authors and Affiliations

  1. Departamento de Microbiología y Genética, Universidad de Salamanca, Calle Zacarías González 2, Lab P1.1, Edificio IBFG, 37007, Salamanca, Spain

    M.-Ángeles Curto, Sandra Moro, Francisco Yanguas & M.-Henar Valdivieso

  2. Instituto de Biología Funcional y Genómica (IBFG), Consejo Superior de Investigaciones Científicas (CSIC), Calle Zacarías González 2, 37007, Salamanca, Spain

    M.-Ángeles Curto, Sandra Moro, Francisco Yanguas, Carmen Gutiérrez-González & M.-Henar Valdivieso

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  1. M.-Ángeles Curto
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  2. Sandra Moro
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Correspondence to M.-Henar Valdivieso.

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Curto, MÁ., Moro, S., Yanguas, F. et al. The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain. Cell. Mol. Life Sci. 75, 1687–1706 (2018). https://doi.org/10.1007/s00018-017-2709-4

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