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

Yuko Fujioka,Nobuo N Noda,Kiyonaga Fujii,Kohki Yoshimoto,Yoshinori Ohsumi,Fuyuhiko Inagaki

doi:10.1074/jbc.m706214200

Yuko Fujioka, Nobuo N Noda + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m706214200

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Abstract

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.

Highlights

Most autophagy-defective mutants display an abnormality in autophagosome formation and cannot survive under starvation conditions
Reconstitution of the AtATG12 Conjugation System in Vitro— In our previous report, we showed that in the plant extracts, AtATG12 was detected as two bands corresponding to the proper molecular weight and a higher molecular weight not detected in homozygous Atatg5-1 or Atatg10-1 mutant plants [20]
We examined whether AtATG5, AtATG7, AtATG10, and AtATG12 represent the minimum components of the AtATG12 conjugation system

Summary

The abbreviations used are

E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; DOPE, dioleoylphosphatidylethanolamine; POPC, 1-palmitoyl-2-oleoylphoshatidylcholine; DOPA, dioleoylphosphatidic acid; PE, phosphatidylethanolamine; HA, hemagglutinin; CBB, Coomassie Brilliant Blue; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry. In Vitro Reconstitution of Plant ATG Conjugation Systems than form I in SDS-PAGE; it was thought to be the PE-conjugated form as in the case of yeast Atg8 [13]. The precise roles of the components of the conjugation systems in the formation of autophagosomes remain unknown. In vivo studies using mammalian and yeast cells have so far supported the presence of cross-talk between the Atg and Atg conjugation systems [15]. In order to clarify the cross-talk mechanism between the AtATG8 and AtATG12 conjugation systems, we reconstituted the AtATG8 and AtATG12 conjugation systems in vitro. Using the in vitro systems, we showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, and AtATG3 inhibited the AtATG12 conjugation system. The reconstitution systems we present allow us to study the relationship between the two conjugation systems and to identify and characterize any other factors that can regulate these conjugation systems

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION