The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy

Abstract

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.

Extracellular matrix regulation of autophagy

“…The same E1-like enzyme, ATG7 and a distinct E2-like enzyme, ATG3, mediates this lipidation. Recent biochemical evidence indicates that the ATG12:5 complex possesses a E3-like activity required for the efficient PE-lipidation of ATG8 [46,47]. The lipid modification of ATG8/LC3 causes it to relocate to autophagosome membrane, serving as useful autophagy marker; indeed, monitoring ATG8/LC3 relocation to puncta or measuring the levels of PE-modified LC3 (LC3-II) using immunoblotting are two methods commonly used to measure autophagosome formation [21].…”

A current perspective of autophagosome biogenesis

“…In addition, the membrane-binding ability of yeast Atg5, which can be inhibited by Atg12 and activated by Atg16, has also been reported [49]. The Atg12-Atg5-Atg16 complex functions as the E3-like enzyme for the other ubiquitin-like conjugation system: the Atg8/LC3 conjugation system [50].…”

“…The C-terminal glycine is conjugated to the amino group of PE, and Atg7 and Atg3 act as the E1-like and E2-like enzymes for this conjugation [52]. The Atg12-Atg5-Atg16 complex acts as the E3-like enzyme [50]. Since the Atg12-Atg5-Atg16(L1) complex accumulates on the isolation membrane and its precursor, Atg8/ LC3-PE conjugation takes place on these membranes.…”

ATG16L1: A multifunctional susceptibility factor in Crohn disease

“…[40][41][42] Moreover, the ATG12-ATG5-ATG16L1 conjugate facilitates the lipidation of LC3-I. 43 Upon autophagosome closure, the LC3-II on the outer surface of the autophagosome is cleaved from PE and released back into the cytosol, along with the other ATG proteins; the LC3-II that is present on the concave surface remains associated with the completed autophagosome. [44][45][46] As a consequence, LC3-II has widely been used as an autophagic marker.…”