Fujioka Yuko, Noda Nobuo N, Fujii Kiyonaga, Yoshimoto Kohki, Ohsumi Yoshinori, Inagaki Fuyuhiko
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-21, W-11, Kita-ku, Sapporo 001-0021, Japan.
J Biol Chem. 2008 Jan 25;283(4):1921-8. doi: 10.1074/jbc.M706214200. Epub 2007 Nov 26.
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.
利用酿酒酵母进行的遗传和生化分析表明，存在两种类泛素缀合系统，即Atg8和Atg12系统，它们在自噬过程中发挥着重要作用，自噬是酵母和哺乳动物中保守的大量降解系统。这些缀合系统在拟南芥中也保守；然而，需要进一步详细研究植物自噬相关（ATG）缀合系统与酵母和哺乳动物中的缀合系统的关系。在此，我们描述了使用纯化的重组蛋白对拟南芥ATG8和ATG12（AtATG8和AtATG12）缀合系统进行的体外重建。AtATG12b以依赖于AtATG7、AtATG10和ATP的方式与AtATG5缀合，而AtATG8a以依赖于AtATG7、AtATG3和ATP的方式与磷脂酰乙醇胺（PE）缀合。其他AtATG8同源物（AtATG8b - 8i）同样与PE缀合。AtATG8缀合物可被AtATG4a和AtATG4b去缀合。这些结果支持了拟南芥中的ATG缀合系统与酵母和哺乳动物中的非常相似的假设。有趣的是，体外分析表明AtATG12 - AtATG5缀合物加速了AtATG8 - PE的形成，而AtATG3抑制了AtATG12 - AtATG5缀合物的形成。本文报道的体外缀合系统将为研究两个缀合系统之间的相互作用机制提供一个工具。
