In vivo and in vitro reconstitution of Atg8 conjugation essential for autophagy.
In an analogous manner to protein ubiquitination, The C terminus of Atg8p, a yeast protein essential for autophagy, conjugates to a head group of phosphatidylethanolamine via an amide bond. Though physiological role of this reaction is assigned to membrane organization during autophagy, its molecular details are still unknown. Here, we show that Escherichia coli cells coexpressed Atg8p, Atg7p (E1), and Atg3p (E2) allowed to form conjugate of Atg8p with endogenous PE. Further, we established an in vitro Atg8p-PE reconstitution system using purified Atg8pG116, Atg7p, Atg3p, and PE-containing liposomes, demonstrating that the Atg7p and the Atg3p are minimal catalysts for Atg8p-PE conjugate reaction. Efficiency of this lipidation reaction depends on the state of the substrate, PE (phospholipid bilayer and its lipid composition). It is also suggested that the lipidation induces a conformational change in the N-terminal region of Atg8p. In vitro system developed here will provide a powerful system for further understanding the precise role of lipidation and interaction of two ubiquitin-like systems essential for autophagy.
Ichimura
Yoshinobu
Y
Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki, 444-8585, Japan.
Imamura
Yuko
Y
Emoto
Kazuo
K
Umeda
Masato
M
Noda
Takeshi
T
Ohsumi
Yoshinori
Y
D001343:Autophagy; D000071193:Autophagy-Related Protein 7; D000071190:Autophagy-Related Protein 8 Family; D015153:Blotting, Western; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D015151:Immunoblotting; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000502:physiology*; D010743:Phospholipids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin / Q000494:pharmacology; D025801:Ubiquitin / Q000737:chemistry
C114830:ATG7 protein, S cerevisiae; C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D008055:Lipids; D008081:Liposomes; D008869:Microtubule-Associated Proteins; D010743:Phospholipids; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D014357:Trypsin; D000071193:Autophagy-Related Protein 7; D005998:Glycine