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Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.

The Tob/BTG family is a group of antiproliferative proteins that contain two highly homologous regions named Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D family of deadenylases. The antiproliferative region of human Tob (residues 1-138) and intact hCaf1 were co-expressed in Escherichia coli, purified and successfully cocrystallized. The crystal belongs to the tetragonal space group I422, with unit-cell parameters a = b = 150.9, c = 113.9 A, and is estimated to contain one heterodimer per asymmetric unit. The crystal diffracted to around 2.6 A resolution.