1887

Abstract

The filamentous fungus is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in , we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed . The biological role of putative orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in , was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between and . Deletion of the Sec2p orthologue in (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in ), did not have an obvious phenotype, while deletion in is lethal. Similarly, deletion of the orthologue of the exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in , although severe growth reduction of was observed. Deletion of , and (encoding SecA, SecH and SsoA the orthologues of Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for , similar to the situation in These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in and .

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2014-02-01
2019-12-06
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References

  1. Bennett J. W., Lasure L. L.. ( 1991;). More Gene Manipulations in Fungi. New York:: Academic Press;.
    [Google Scholar]
  2. Bock J. B., Matern H. T., Peden A. A., Scheller R. H.. ( 2001;). A genomic perspective on membrane compartment organization. . Nature 409:, 839–841. [CrossRef][PubMed]
    [Google Scholar]
  3. Bonifacino J. S., Glick B. S.. ( 2004;). The mechanisms of vesicle budding and fusion. . Cell 116:, 153–166. [CrossRef][PubMed]
    [Google Scholar]
  4. Bos C. J., Debets A. J., Swart K., Huybers A., Kobus G., Slakhorst S. M.. ( 1988;). Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger.. Curr Genet 14:, 437–443. [CrossRef][PubMed]
    [Google Scholar]
  5. Carvalho N. D. S. P., Arentshorst M., Jin Kwon M., Meyer V., Ram A. F. J.. ( 2010;). Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. . Appl Microbiol Biotechnol 87:, 1463–1473. [CrossRef][PubMed]
    [Google Scholar]
  6. Carvalho N. D. S. P., Arentshorst M., Weenink X. O., Punt P. J., Van den Hondel C. A. M. J. J., Ram A. F. J.. ( 2011;). Functional YFP-tagging of the essential GDP-mannose transporter reveals an important role for the secretion related small GTPase SrgC protein in maintenance of Golgi bodies in Aspergillus niger.. Fungal Biol 115:, 253–264. [CrossRef][PubMed]
    [Google Scholar]
  7. Carvalho N. D. S. P., Jørgensen T. R., Arentshorst M., Nitsche B. M., Van den Hondel C. A., Archer D. B., Ram A. F. J.. ( 2012;). Genome-wide expression analysis upon constitutive activation of the HacA bZIP transcription factor in Aspergillus niger reveals a coordinated cellular response to counteract ER stress. . BMC Genomics 13:, 350. [CrossRef][PubMed]
    [Google Scholar]
  8. Chen Y. A., Scheller R. H.. ( 2001;). SNARE-mediated membrane fusion. . Nat Rev Mol Cell Biol 2:, 98–106. [CrossRef][PubMed]
    [Google Scholar]
  9. de Ruiter-Jacobs Y. M. J. T., Broekhuijsen M., Unkles S. E., Campbell E. I., Kinghorn J. R., Contreras R., Pouwels P. H., Van den Hondel C. A. M. J. J.. ( 1989;). A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae.. Curr Genet 16:, 159–163. [CrossRef][PubMed]
    [Google Scholar]
  10. Fdez E., Martínez-Salvador M., Beard M., Woodman P., Hilfiker S.. ( 2010;). Transmembrane-domain determinants for SNARE-mediated membrane fusion. . J Cell Sci 123:, 2473–2480. [CrossRef][PubMed]
    [Google Scholar]
  11. Fleissner A., Dersch P.. ( 2010;). Expression and export: recombinant protein production systems for Aspergillus.. Appl Microbiol Biotechnol 87:, 1255–1270. [CrossRef][PubMed]
    [Google Scholar]
  12. Furuta N., Fujimura-Kamada K., Saito K., Yamamoto T., Tanaka K.. ( 2007;). Endocytic recycling in yeast is regulated by putative phospholipid translocases and the Ypt31p/32p-Rcy1p pathway. . Mol Biol Cell 18:, 295–312. [CrossRef][PubMed]
    [Google Scholar]
  13. Grote E., Baba M., Ohsumi Y., Novick P. J.. ( 2000;). Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion. . J Cell Biol 151:, 453–466. [CrossRef][PubMed]
    [Google Scholar]
  14. Gupta G. D., Heath I. B.. ( 2002;). Predicting the distribution, conservation, and functions of SNAREs and related proteins in fungi. . Fungal Genet Biol 36:, 1–21. [CrossRef][PubMed]
    [Google Scholar]
  15. Harris S. D., Read N. D., Roberson R. W., Shaw B., Seiler S., Plamann M., Momany M.. ( 2005;). Polarisome meets Spitzenkörper: microscopy, genetics, and genomics converge. . Eukaryot Cell 4:, 225–229. [CrossRef][PubMed]
    [Google Scholar]
  16. Harsay E., Bretscher A.. ( 1995;). Parallel secretory pathways to the cell surface in yeast. . J Cell Biol 131:, 297–310. [CrossRef][PubMed]
    [Google Scholar]
  17. Hayakawa Y., Ishikawa E., Shoji J. Y., Nakano H., Kitamoto K.. ( 2011;). Septum-directed secretion in the filamentous fungus Aspergillus oryzae.. Mol Microbiol 81:, 40–55. [CrossRef][PubMed]
    [Google Scholar]
  18. Heider M. R., Munson M.. ( 2012;). Exorcising the exocyst complex. . Traffic 13:, 898–907. [CrossRef][PubMed]
    [Google Scholar]
  19. Itzen A., Rak A., Goody R. S.. ( 2007;). Sec2 is a highly efficient exchange factor for the Rab protein Sec4. . J Mol Biol 365:, 1359–1367. [CrossRef][PubMed]
    [Google Scholar]
  20. Jacobs D. I., Olsthoorn M. M. A, Maillet I., Akeroyd M., Breestraat S., Donkers S., Van der Hoeven R. A. M., Van den Hondel C. A. M. J. J, Kooistra R.. & other authors ( 2009;). Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics. . Fungal Genet Biol 46: (Suppl. 1), S141–S152. [CrossRef][PubMed]
    [Google Scholar]
  21. Jäntti J., Aalto M. K., Oyen M., Sundqvist L., Keränen S., Ronne H.. ( 2002;). Characterization of temperature-sensitive mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and evidence of a distinct function for Sso1p in sporulation. . J Cell Sci 115:, 409–420.[PubMed]
    [Google Scholar]
  22. 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. [CrossRef][PubMed]
    [Google Scholar]
  23. Jørgensen T. R., Goosen T., Van der Hondel C. A. M. J. J., Ram A. F. J., Iversen J. J. L.. ( 2009;). Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway. . BMC Genomics 10:, 44. [CrossRef][PubMed]
    [Google Scholar]
  24. Jørgensen T. R., Nitsche B. M., Lamers G. E., Arentshorst M., Van den Hondel C. A., Ram A. F.. ( 2010;). Transcriptomic insights into the physiology of Aspergillus niger approaching a specific growth rate of zero. . Appl Environ Microbiol 76:, 5344–5355. [CrossRef][PubMed]
    [Google Scholar]
  25. Kienle N., Kloepper T. H., Fasshauer D.. ( 2009;). Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi. . BMC Evol Biol 9:, 19. [CrossRef][PubMed]
    [Google Scholar]
  26. Kuratsu M., Taura A., Shoji J. Y., Kikuchi S., Arioka M., Kitamoto K.. ( 2007;). Systematic analysis of SNARE localization in the filamentous fungus Aspergillus oryzae.. Fungal Genet Biol 44:, 1310–1323. [CrossRef][PubMed]
    [Google Scholar]
  27. Kwon M. J., Arentshorst M., Roos E. D., Van den Hondel C. A., Meyer V., Ram A. F.. ( 2011;). Functional characterization of Rho GTPases in Aspergillus niger uncovers conserved and diverged roles of Rho proteins within filamentous fungi. . Mol Microbiol 79:, 1151–1167. [CrossRef][PubMed]
    [Google Scholar]
  28. Kwon M. J., Jørgensen T. R., Nitsche B. M., Arentshorst M., Park J., Ram A. F., Meyer V.. ( 2012;). The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger.. BMC Genomics 13:, 701. [CrossRef][PubMed]
    [Google Scholar]
  29. Kwon M. J., Nitsche B. M., Arentshorst M., Jørgensen T. R., Ram A. F. J., Meyer V.. ( 2013;). The transcriptomic signature of RacA activation and inactivation provides new insights into the morphogenetic network of Aspergillus niger.. PLoS ONE 8:, e68946. [CrossRef][PubMed]
    [Google Scholar]
  30. Langosch D., Hofmann M., Ungermann C.. ( 2007;). The role of transmembrane domains in membrane fusion. . Cell Mol Life Sci 64:, 850–864. [CrossRef][PubMed]
    [Google Scholar]
  31. Leucci M. R., Di Sansebastiano G. P., Gigante M., Dalessandro G., Piro G.. ( 2007;). Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways. . Planta 225:, 1001–1017. [CrossRef][PubMed]
    [Google Scholar]
  32. Li Z., Vizeacoumar F. J., Bahr S., Li J., Warringer J., Vizeacoumar F. S., Min R., Vandersluis B., Bellay J.. & other authors ( 2011;). Systematic exploration of essential yeast gene function with temperature-sensitive mutants. . Nat Biotechnol 29:, 361–367. [CrossRef][PubMed]
    [Google Scholar]
  33. Lu X., Zhang Y., Shin Y. K.. ( 2008;). Supramolecular SNARE assembly precedes hemifusion in SNARE-mediated membrane fusion. . Nat Struct Mol Biol 15:, 700–706. [CrossRef][PubMed]
    [Google Scholar]
  34. Meyer V.. ( 2008;). Genetic engineering of filamentous fungi – progress, obstacles and future trends. . Biotechnol Adv 26:, 177–185. [CrossRef][PubMed]
    [Google Scholar]
  35. Meyer V., Arentshorst M., El-Ghezal A., Drews A. C., Kooistra R., Van den Hondel C. A., Ram A. F.. ( 2007a;). Highly efficient gene targeting in the Aspergillus niger kusA mutant. . J Biotechnol 128:, 770–775. [CrossRef][PubMed]
    [Google Scholar]
  36. Meyer V., Damveld R. A., Arentshorst M., Stahl U., Van den Hondel C. A., Ram A. F.. ( 2007b;). Survival in the presence of antifungals: genome-wide expression profiling of Aspergillus niger in response to sublethal concentrations of caspofungin and fenpropimorph. . J Biol Chem 282:, 32935–32948. [CrossRef][PubMed]
    [Google Scholar]
  37. Meyer V., Ram A. F., Punt P. J.. ( 2010;). Genetics, genetic manipulation and approaches to strain improvement of filamentous fungi. . In Manual of Industrial Microbiology and Biotechnology, pp. 318–329. Edited by Balz R. H., Davies J. E., Demain A. L... Washington, DC:: ASM;.
    [Google Scholar]
  38. Meyer V., Wanka F., Van Gent J., Arentshorst M., Van den Hondel C. A., Ram A. F.. ( 2011a;). Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger.. Appl Environ Microbiol 77:, 2975–2983. [CrossRef][PubMed]
    [Google Scholar]
  39. Meyer V., Wu B., Ram A. F.. ( 2011b;). Aspergillus as a multi-purpose cell factory: current status and perspectives. . Biotechnol Lett 33:, 469–476. [CrossRef][PubMed]
    [Google Scholar]
  40. Nichols B. J., Ungermann C., Pelham H. R., Wickner W. T., Haas A.. ( 1997;). Homotypic vacuolar fusion mediated by t- and v-SNAREs. . Nature 387:, 199–202. [CrossRef][PubMed]
    [Google Scholar]
  41. Novick P., Field C., Schekman R.. ( 1980;). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. . Cell 21:, 205–215. [CrossRef][PubMed]
    [Google Scholar]
  42. Pel H. J., de Winde J. H., Archer D. B., Dyer P. S., Hofmann G., Schaap P. J., Turner G., de Vries R. P., Albang R.. & other authors ( 2007;). Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. . Nat Biotechnol 25:, 221–231. [CrossRef][PubMed]
    [Google Scholar]
  43. Protopopov V., Govindan B., Novick P., Gerst J. E.. ( 1993;). Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae.. Cell 74:, 855–861. [CrossRef][PubMed]
    [Google Scholar]
  44. Punt P. J., Oliver R. P., Dingemanse M. A., Pouwels P. H., Van den Hondel C. A.. ( 1987;). Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli.. Gene 56:, 117–124. [CrossRef][PubMed]
    [Google Scholar]
  45. Punt P. J., Seiboth B., Weenink X. O., Van Zeijl C., Lenders M., Konetschny C., Ram A. F., Montijn R., Kubicek C. P., Van den Hondel C. A.. ( 2001;). Identification and characterization of a family of secretion-related small GTPase-encoding genes from the filamentous fungus Aspergillus niger: a putative SEC4 homologue is not essential for growth. . Mol Microbiol 41:, 513–525. [CrossRef][PubMed]
    [Google Scholar]
  46. Roca M. G., Kuo H. C., Lichius A., Freitag M., Read N. D.. ( 2010;). Nuclear dynamics, mitosis, and the cytoskeleton during the early stages of colony initiation in Neurospora crassa.. Eukaryot Cell 9:, 1171–1183. [CrossRef][PubMed]
    [Google Scholar]
  47. Sagt C. M. J., ten Haaft P. J., Minneboo I. M., Hartog M. P., Damveld R. A., Van der Laan J. M., Akeroyd M., Wenzel T. J., Luesken F. A.. & other authors ( 2009;). Peroxicretion: a novel secretion pathway in the eukaryotic cell. . BMC Biotechnol 9:, 48. [CrossRef][PubMed]
    [Google Scholar]
  48. Saloheimo M., Pakula T. M.. ( 2012;). The cargo and the transport system: secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina).. Microbiology 158:, 46–57. [CrossRef][PubMed]
    [Google Scholar]
  49. Sambrook J., Russell D. W.. ( 2001;). Molecular Cloning: a Laboratory Manual, , 3rd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  50. Schekman R.. ( 2010;). Charting the secretory pathway in a simple eukaryote. . Mol Biol Cell 21:, 3781–3784. [CrossRef][PubMed]
    [Google Scholar]
  51. Segev N.. ( 2001a;). Ypt and Rab GTPases: insight into functions through novel interactions. . Curr Opin Cell Biol 13:, 500–511. [CrossRef][PubMed]
    [Google Scholar]
  52. Segev N.. ( 2001b;). Ypt/Rab GTPases: regulators of protein trafficking. . Sci STKE 2001:, re11.[PubMed]
    [Google Scholar]
  53. Steinberg G.. ( 2007;). Hyphal growth: a tale of motors, lipids, and the Spitzenkörper. . Eukaryot Cell 6:, 351–360. [CrossRef][PubMed]
    [Google Scholar]
  54. Taheri-Talesh N., Horio T., Araujo-Bazán L., Dou X., Espeso E. A., Peñalva M. A., Osmani S. A., Oakley B. R.. ( 2008;). The tip growth apparatus of Aspergillus nidulans.. Mol Biol Cell 19:, 1439–1449. [CrossRef][PubMed]
    [Google Scholar]
  55. Taheri-Talesh N., Xiong Y., Oakley B. R.. ( 2012;). The functions of myosin II and myosin V homologs in tip growth and septation in Aspergillus nidulans.. PLoS ONE 7:, e31218. [CrossRef][PubMed]
    [Google Scholar]
  56. Titorenko V. I., Ogrydziak D. M., Rachubinski R. A.. ( 1997;). Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.. Mol Cell Biol 17:, 5210–5226.[PubMed]
    [Google Scholar]
  57. Ungar D., Hughson F. M.. ( 2003;). SNARE protein structure and function. . Annu Rev Cell Dev Biol 19:, 493–517. [CrossRef][PubMed]
    [Google Scholar]
  58. Valkonen M., Kalkman E. R., Saloheimo M., Penttilä M., Read N. D., Duncan R. R.. ( 2007;). Spatially segregated SNARE protein interactions in living fungal cells. . J Biol Chem 282:, 22775–22785. [CrossRef][PubMed]
    [Google Scholar]
  59. Van Hartingsveldt W., Mattern I. E., Van Zeijl C. M. J., Pouwels P. H., Van den Hondel C. A. M. J. J.. ( 1987;). Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. . Mol Gen Genet 206:, 71–75. [CrossRef][PubMed]
    [Google Scholar]
  60. Varadarajan R., Nagarajaram H. A., Ramakrishnan C.. ( 1996;). A procedure for the prediction of temperature-sensitive mutants of a globular protein based solely on the amino acid sequence. . Proc Natl Acad Sci U S A 93:, 13908–13913. [CrossRef][PubMed]
    [Google Scholar]
  61. Walch-Solimena C., Collins R. N., Novick P. J.. ( 1997;). Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles. . J Cell Biol 137:, 1495–1509. [CrossRef][PubMed]
    [Google Scholar]
  62. Wen W., Chen L., Wu H., Sun X., Zhang M., Banfield D. K.. ( 2006;). Identification of the yeast R-SNARE Nyv1p as a novel longin domain-containing protein. . Mol Biol Cell 17:, 4282–4299. [CrossRef][PubMed]
    [Google Scholar]
  63. Yoshimori T., Keller P., Roth M. G., Simons K.. ( 1996;). Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. . J Cell Biol 133:, 247–256. [CrossRef][PubMed]
    [Google Scholar]
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