In vitro reconstitution of plant ATG8 and ATG12 conjugation systems essential for autophagy

doi:10.1074/jbc.M706214200

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Papers In Press, published online ahead of print November 26, 2007
J. Biol. Chem, 10.1074/jbc.M706214200
Submitted on July 27, 2007
Revised on October 29, 2007
Accepted on November 26, 2007

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 Inagaki

Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812

Corresponding Author: finagaki{at}pharm.hokudai.ac.jp

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 conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana (At) ATG8 and ATG12 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-i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results show 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 crosstalk mechanism between two conjugation systems.

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