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J. Biol. Chem., Vol. 283, Issue 4, 1921-1928, January 25, 2008

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In Vitro Reconstitution of Plant Atg8 and Atg12 Conjugation Systems Essential for Autophagy^*

Yuko Fujioka, Nobuo N. Noda, Kiyonaga Fujii, Kohki Yoshimoto, Yoshinori Ohsumi, and Fuyuhiko Inagaki¹

From the Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-21, W-11, Kita-ku, Sapporo 001-0021, Japan and the Division of Molecular Cell Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan

Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b–8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.

Received for publication, July 27, 2007 , and in revised form, October 29, 2007.

^* This work was supported by Grant-in-Aids for priority areas and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. This work was carried out under NIBB Cooperative Research Program 4-148. 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 Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-21, W-11, Kita-ku, Sapporo 001-0021, Japan. Tel.: 81-11-706-9011; Fax: 81-11-706-9012; E-mail: finagaki{at}pharm.hokudai.ac.jp.

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