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J. Biol. Chem., Vol. 281, Issue 6, 3017-3024, February 10, 2006

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Phosphatidylserine in Addition to Phosphatidylethanolamine Is an in Vitro Target of the Mammalian Atg8 Modifiers, LC3, GABARAP, and GATE-16^*

From the Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan

In yeast, phosphatidylethanolamine is a target of the Atg8 modifier in ubiquitylation-like reactions essential for autophagy. Three human Atg8 (hAtg8) homologs, LC3, GABARAP, and GATE-16, have been characterized as modifiers in reactions mediated by hAtg7 (an E1-like enzyme) and hAtg3 (an E2-like enzyme) as in yeast Atg8 lipidation, but their final targets have not been identified. The results of a recent study in which COS7 cells were incubated with [¹⁴C]ethanolamine for 48 h suggested that phosphatidylethanolamine is a target of LC3. However, these results were not conclusive because of the long incubation time. To identify the phospholipid targets of Atg8 homologs, we reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. Each purified mutant Atg8 homolog with an exposed C-terminal Gly formed an E1-substrate intermediate with hAtg7 via a thioester bond in an ATP-dependent manner and formed an E2-substrate intermediate with hAtg3 via a thioester bond dependent on ATP and hAtg7. A conjugated form of each Atg8 homolog was observed in the presence of hAtg7, hAtg3, ATP, and liposomes. In addition to phosphatidylethanolamine, in vitro conjugation experiments using synthetic phospholipid liposomes showed that phosphatidylserine is also a target of LC3, GABARAP, and GATE-16. In contrast, thin layer chromatography of phospholipids released on hAtg4B-digestion from endogenous LC3-phospholipid conjugate revealed that phosphatidylethanolamine, but not phosphatidylserine, is the predominant target phospholipid of LC3 in vivo. The discrepancy between in vitro and in vivo reactions suggested that there may be selective factor(s) involved in the endogenous LC3 conjugation system.

Received for publication, May 31, 2005 , and in revised form, October 18, 2005.

^* This work was supported in part by Ministry of Education, Science, Sports, and Culture of Japan Grants-in-aid for Scientific Research 15590254 (to I. T.), 09680629 (to T. U.), and 12470040 (to E. K.); Grant-in-aid for Scientific Research on Priority Areas 12146205 (to E. K.); and a grant (to E. K.) from the Science Research Promotion Fund of the Japan Private School Promotion Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: 81-3-5802-1031; Fax: 81-3-5802-5889; E-mail: kominami{at}med.juntendo.ac.jp.

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