We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.
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18 October 1999
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October 18 1999
Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast
Takayoshi Kirisako,
Takayoshi Kirisako
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
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Misuzu Baba,
Misuzu Baba
bDepartment of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
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Naotada Ishihara,
Naotada Ishihara
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
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Kouichi Miyazawa,
Kouichi Miyazawa
cDepartment of Biosciences, Teikyo University of Science and Technology, Yamanashi 409-0193, Japan
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Mariko Ohsumi,
Mariko Ohsumi
cDepartment of Biosciences, Teikyo University of Science and Technology, Yamanashi 409-0193, Japan
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Tamotsu Yoshimori,
Tamotsu Yoshimori
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
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Takeshi Noda,
Takeshi Noda
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
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Yoshinori Ohsumi
Yoshinori Ohsumi
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
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Takayoshi Kirisako
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
Misuzu Baba
bDepartment of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
Naotada Ishihara
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
Kouichi Miyazawa
cDepartment of Biosciences, Teikyo University of Science and Technology, Yamanashi 409-0193, Japan
Mariko Ohsumi
cDepartment of Biosciences, Teikyo University of Science and Technology, Yamanashi 409-0193, Japan
Tamotsu Yoshimori
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
Takeshi Noda
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
Yoshinori Ohsumi
aDepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
1.used in this paper: ALP, alkaline phosphatase; API, aminopeptidase I; HA, hemagglutinin; ORF, open reading frame; proAPI, precursor form of aminopeptidase I
Received:
July 07 1999
Revision Requested:
August 25 1999
Accepted:
September 07 1999
Online ISSN: 1540-8140
Print ISSN: 0021-9525
© 1999 The Rockefeller University Press
1999
The Rockefeller University Press
J Cell Biol (1999) 147 (2): 435–446.
Article history
Received:
July 07 1999
Revision Requested:
August 25 1999
Accepted:
September 07 1999
Citation
Takayoshi Kirisako, Misuzu Baba, Naotada Ishihara, Kouichi Miyazawa, Mariko Ohsumi, Tamotsu Yoshimori, Takeshi Noda, Yoshinori Ohsumi; Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast. J Cell Biol 18 October 1999; 147 (2): 435–446. doi: https://doi.org/10.1083/jcb.147.2.435
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