1887

Abstract

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome–vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast . Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the Δ and Δ mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation.

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2009-12-01
2024-03-28
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References

  1. Alfa C., Fantes P., Hyams J. S., Mcleod M., Warbrick E. 1993 Experiments with Fission Yeast: a Laboratory Course Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  2. Bahler J., Wu J. Q., Longtine M. S., Shah N. G., McKenzie A. III, Steever A. B., Wach A., Philippsen P., Pringle J. R. 1998; Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe . Yeast 14:943–951
    [Google Scholar]
  3. Chan T. F., Bertram P. G., Ai W., Zheng X. F. 2001; Regulation of APG14 expression by the GATA-type transcription factor Gln3p. J Biol Chem 276:6463–6467
    [Google Scholar]
  4. Chardwiriyapreecha S., Shimazu M., Morita T., Sekito T., Akiyama K., Takegawa K., Kakinuma Y. 2008; Identification of the fnx1 + and fnx2 + genes for vacuolar amino acid transporters in Schizosaccharomyces pombe . FEBS Lett 582:2225–2230
    [Google Scholar]
  5. Cheong H., Yorimitsu T., Reggiori F., Legakis J. E., Wang C. W., Klionsky D. J. 2005; Atg17 regulates the magnitude of the autophagic response. Mol Biol Cell 16:3438–3453
    [Google Scholar]
  6. Chu S., DeRisi J., Eisen M., Mulholland J., Botstein D., Brown P. O., Herskowitz I. 1998; The transcriptional program of sporulation in budding yeast. Science 282:699–705
    [Google Scholar]
  7. Deutschbauer A. M., Williams R. M., Chu A. M., Davis R. W. 2002; Parallel phenotypic analysis of sporulation and postgermination growth in Saccharomyces cerevisiae . Proc Natl Acad Sci U S A 99:15530–15535
    [Google Scholar]
  8. Funakoshi T., Matsuura A., Noda T., Ohsumi Y. 1997; Analyses of APG13 gene involved in autophagy in yeast, Saccharomyces cerevisiae . Gene 192:207–213
    [Google Scholar]
  9. Hagan I. M., Hyams J. S. 1988; The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe . J Cell Sci 89:343–357
    [Google Scholar]
  10. Helliwell S. B., Wagner P., Kunz J., Deuter-Reinhard M., Henriquez R., Hall M. N. 1994; TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol Biol Cell 5:105–118
    [Google Scholar]
  11. Hosokawa N., Hara Y., Mizushima N. 2006; Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett 580:2623–2629
    [Google Scholar]
  12. Ichimura Y., Kirisako T., Takao T., Satomi Y., Shimonishi Y., Ishihara N., Mizushima N., Tanida I., Kominami E. other authors 2000; A ubiquitin-like system mediates protein lipidation. Nature 408:488–492
    [Google Scholar]
  13. Isshiki T., Mochizuki N., Maeda T., Yamamoto M. 1992; Characterization of a fission yeast gene, gpa2, that encodes a G alpha subunit involved in the monitoring of nutrition. Genes Dev 6:2455–2462
    [Google Scholar]
  14. Kamada Y., Funakoshi T., Shintani T., Nagano K., Ohsumi M., Ohsumi Y. 2000; Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J Cell Biol 150:1507–1513
    [Google Scholar]
  15. Kirisako T., Baba M., Ishihara N., Miyazawa K., Ohsumi M., Yoshimori T., Noda T., Ohsumi Y. 1999; Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J Cell Biol 147:435–446
    [Google Scholar]
  16. Kirisako T., Ichimura Y., Okada H., Kabeya Y., Mizushima N., Yoshimori T., Ohsumi M., Takao T., Noda T., Ohsumi Y. 2000; The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol 151:263–276
    [Google Scholar]
  17. Klionsky D. J., Cuervo A. M., Seglen P. O. 2007; Methods for monitoring autophagy from yeast to human. Autophagy 3:181–206
    [Google Scholar]
  18. Kohda T. A., Tanaka K., Konomi M., Sato M., Osumi M., Yamamoto M. 2007; Fission yeast autophagy induced by nitrogen starvation generates a nitrogen source that drives adaptation processes. Genes Cells 12:155–170
    [Google Scholar]
  19. Maeda T., Mochizuki N., Yamamoto M. 1990; Adenylyl cyclase is dispensable for vegetative cell growth in the fission yeast Schizosaccharomyces pombe . Proc Natl Acad Sci U S A 87:7814–7818
    [Google Scholar]
  20. Maeda T., Watanabe Y., Kunitomo H., Yamamoto M. 1994; Cloning of the pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe . J Biol Chem 269:9632–9637
    [Google Scholar]
  21. Mata J., Lyne R., Burns G., Bahler J. 2002; The transcriptional program of meiosis and sporulation in fission yeast. Nat Genet 32:143–147
    [Google Scholar]
  22. Matsuura A., Tsukada M., Wada Y., Ohsumi Y. 1997; Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae . Gene 192:245–250
    [Google Scholar]
  23. Mizushima N., Noda T., Yoshimori T., Tanaka Y., Ishii T., George M. D., Klionsky D. J., Ohsumi M., Ohsumi Y. 1998; A protein conjugation system essential for autophagy. Nature 395:395–398
    [Google Scholar]
  24. Moreno S., Klar A., Nurse P. 1991; Molecular genetic analysis of fission yeast Schizosaccharomyces pombe . Methods Enzymol 194:795–823
    [Google Scholar]
  25. Morishita M., Morimoto F., Kitamura K., Koga T., Fukui Y., Maekawa H., Yamashita I., Shimoda C. 2002; Phosphatidylinositol 3-phosphate 5-kinase is required for the cellular response to nutritional starvation and mating pheromone signals in Schizosaccharomyces pombe . Genes Cells 7:199–215
    [Google Scholar]
  26. Morita T., Takegawa K. 2004; A simple and efficient procedure for transformation of Schizosaccharomyces pombe . Yeast 21:613–617
    [Google Scholar]
  27. Mukaiyama H., Oku M., Baba M., Samizo T., Hammond A. T., Glick B. S., Kato N., Sakai Y. 2002; Paz2 and 13 other PAZ gene products regulate vacuolar engulfment of peroxisomes during micropexophagy. Genes Cells 7:75–90
    [Google Scholar]
  28. Nakamura T., Nakamura-Kubo M., Hirata A., Shimoda C. 2001; The Schizosaccharomyces pombe spo3 + gene is required for assembly of the forespore membrane and genetically interacts with psy1 +-encoding syntaxin-like protein. Mol Biol Cell 12:3955–3972
    [Google Scholar]
  29. Nakamura T., Asakawa H., Nakase Y., Kashiwazaki J., Hiraoka Y., Shimoda C. 2008; Live observation of forespore membrane formation in fission yeast. Mol Biol Cell 19:3544–3553
    [Google Scholar]
  30. Nakashima A., Hasegawa T., Mori S., Ueno M., Tanaka S., Ushimaru T., Sato S., Uritani M. 2006; A starvation-specific serine protease gene, isp6 +, is involved in both autophagy and sexual development in Schizosaccharomyces pombe . Curr Genet 49:403–413
    [Google Scholar]
  31. Noda T., Ohsumi Y. 1998; Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J Biol Chem 273:3963–3966
    [Google Scholar]
  32. Ohsumi Y. 2001; Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol 2:211–216
    [Google Scholar]
  33. Okazaki K., Okazaki N., Kume K., Jinno S., Tanaka K., Okayama H. 1990; High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe . Nucleic Acids Res 18:6485–6489
    [Google Scholar]
  34. Onodera J., Ohsumi Y. 2005; Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation. J Biol Chem 280:31582–31586
    [Google Scholar]
  35. Primig M., Williams R. M., Winzeler E. A., Tevzadze G. G., Conway A. R., Hwang S. Y., Davis R. W., Esposito R. E. 2000; The core meiotic transcriptome in budding yeasts. Nat Genet 26:415–423
    [Google Scholar]
  36. Richie D. L., Fuller K. K., Fortwendel J., Miley M. D., McCarthy J. W., Feldmesser M., Rhodes J. C., Askew D. S. 2007; Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus . Eukaryot Cell 6:2437–2447
    [Google Scholar]
  37. Russnak R., Konczal D., McIntire S. L. 2001; A family of yeast proteins mediating bidirectional vacuolar amino acid transport. J Biol Chem 276:23849–23857
    [Google Scholar]
  38. Shintani T., Klionsky D. J. 2004; Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway. J Biol Chem 279:29889–29894
    [Google Scholar]
  39. Straub M., Bredschneider M., Thumm M. 1997; AUT3, a serine/threonine kinase gene, is essential for autophagocytosis in Saccharomyces cerevisiae . J Bacteriol 179:3875–3883
    [Google Scholar]
  40. Suga M., Hatakeyama T. 2001; High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation. Yeast 18:1015–1021
    [Google Scholar]
  41. Suzuki K., Kirisako T., Kamada Y., Mizushima N., Noda T., Ohsumi Y. 2001; The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J 20:5971–5981
    [Google Scholar]
  42. Takeshige K., Baba M., Tsuboi S., Noda T., Ohsumi Y. 1992; Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol 119:301–311
    [Google Scholar]
  43. Tsukada M., Ohsumi Y. 1993; Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae . FEBS Lett 333:169–174
    [Google Scholar]
  44. Uritani M., Hidaka H., Hotta Y., Ueno M., Ushimaru T., Toda T. 2006; Fission yeast Tor2 links nitrogen signals to cell proliferation and acts downstream of the Rheb GTPase. Genes Cells 11:1367–1379
    [Google Scholar]
  45. Weisman R. 2004; The fission yeast TOR proteins and the rapamycin response: an unexpected tale. Curr Top Microbiol Immunol 279:85–95
    [Google Scholar]
  46. Weisman R., Choder M., Koltin Y. 1997; Rapamycin specifically interferes with the developmental response of fission yeast to starvation. J Bacteriol 179:6325–6334
    [Google Scholar]
  47. Weisman R., Finkelstein S., Choder M. 2001; Rapamycin blocks sexual development in fission yeast through inhibition of the cellular function of an FKBP12 homolog. J Biol Chem 276:24736–24742
    [Google Scholar]
  48. Welton R. M., Hoffman C. S. 2000; Glucose monitoring in fission yeast via the Gpa2 galpha, the git5 Gbeta and the git3 putative glucose receptor. Genetics 156:513–521
    [Google Scholar]
  49. Woods A., Sherwin T., Sasse R., MacRae T. H., Baines A. J., Gull K. 1989; Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci 93:491–500
    [Google Scholar]
  50. Yang Z., Huang J., Geng J., Nair U., Klionsky D. J. 2006; Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Mol Biol Cell 17:5094–5104
    [Google Scholar]
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