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Published online 12 February 2001. doi:10.1083/jcb.152.4.657
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© The Rockefeller University Press, 0021-9525/2001/2/657/ $5.00
The Journal of Cell Biology, Volume 152, Number 4, February 19, 2001 657-668

Original Article

Dissection of Autophagosome Formation using Apg5-deficient Mouse Embryonic Stem Cells

Noboru Mizushimaa,b, Akitsugu Yamamotoc, Masahiko Hatanod, Yoshinori Kobayashib,e, Yukiko Kabeyab, Kuninori Suzukib,e, Takeshi Tokuhisad, Yoshinori Ohsumib,e, and Tamotsu Yoshimorib,e
a Unit Process and Combined Circuit, PRESTO, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan
b Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
c Department of Physiology, Kansai Medical University, Moriguchi 570-8506, Japan
d Department of Developmental Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
e Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan

Correspondence to: Noboru Mizushima, Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. Tel:81-564-55-7517 Fax:81-564-55-7516 E-mail:nmizu{at}nibb.ac.jp.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein–tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

Key Words: autophagy, ubiquitin-like protein, autophagosome, isolation membrane, gene targeting


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