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Autophagy. 2013 Oct;9(10):1491-9. doi: 10.4161/auto.25529. Epub 2013 Jul 10.

Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site.

Author information

1
Department of Physiology and Cell Biology; Tokyo Medical and Dental University; Bunkyo-ku, Tokyo Japan; Department of Biochemistry and Molecular Biology; Graduate School and Faculty of Medicine; The University of Tokyo; Bunkyo-ku, Tokyo Japan.
2
Department of Physiology and Cell Biology; Tokyo Medical and Dental University; Bunkyo-ku, Tokyo Japan.
3
Institute for Integrated Cell-Material Sciences (WPI-iCeMS); Kyoto University; Sakyo-ku, Kyoto Japan.

Abstract

Autophagosome formation is governed by sequential functions of autophagy-related (ATG) proteins. Although their genetic hierarchy in terms of localization to the autophagosome formation site has been determined, their temporal relationships remain largely unknown. In this study, we comprehensively analyzed the recruitment of mammalian ATG proteins to the autophagosome formation site by live-cell imaging, and determined their temporal relationships. Although ULK1 and ATG5 are separated in the genetic hierarchy, they synchronously accumulate at pre-existing VMP1-positive punctate structures, followed by recruitment of ATG14, ZFYVE1, and WIPI1. Only a small number of ATG9 vesicles appear to be associated with these structures. Finally, LC3 and SQSTM1/p62 accumulate synchronously, while the other ATG proteins dissociate from the autophagic structures. These results suggest that autophagosome formation takes place on the VMP1-containing domain of the endoplasmic reticulum or a closely related structure, where ULK1 and ATG5 complexes are synchronously recruited.

KEYWORDS:

ATG5; ULK1; VMP1; autophagosome; live-cell imaging

PMID:
23884233
DOI:
10.4161/auto.25529
[Indexed for MEDLINE]

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