The Crystal Structure of Atg3, an Autophagy-related Ubiquitin Carrier Protein (E2) Enzyme that Mediates Atg8 Lipidation

Abstract

Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-Å resolution. Atg3 has an ␣/␤-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of ϳ80 residues and has a random coil structure in solution and another with a long ␣-helical structure that protrudes from the core region as far as 30 Å . In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.

The in vitro cleavage of the hAtg proteins by cell death proteases

“…Furthermore the cleavage of Atg3 by calpain 1 in the region separating the domains that interact with Atg7 and Atg8 may result in uncoordinated conjugate formation and inefficient autophagy induction. 36 This site is also conserved in the mouse and rat. Further study will be required to ascertain whether Atg3 cleavage has any cellular relevance.…”

“…35 The cleavage sites of caspase-3, -6 or -8 in Atg3 are predicted to fall in the region that is required for its interaction with Atg7, 36 ( Table 1) and are conserved in the mouse and rat. Atg7 and Atg3 are the E1 and E2 enzymes respectively that catalyse the lipidation of Atg8 (LC3, GATE-16 or GABARAP).…”

Visualization of Atg3 during Autophagosome Formation in Saccharomyces cerevisiae

“…We concluded that a GFP tag at either terminus of Atg3 yielded a non-functional protein that would be inappropriate for use in this study. Therefore, we screened for permissive sites for GFP insertion, referring to structural information obtained previously for S. cerevisiae Atg3 (20). In particular, we evaluated three GFP insertion sites C-terminal to the handle region (Fig.…”

“…GFP tagging at the N terminus might obstruct the interaction of this domain with PE, resulting in functional impairment (32). Although it is not clear whether the C-terminal domain of Atg3 has an interaction partner, this domain is located very close to the E2-like domain of Atg3 (20). In the Atg3⅐Atg7 complex, the flexible region of Atg3 is rearranged to interact with a distal groove in the N-terminal domain of Atg7 (33).…”

The machinery of macroautophagy

“…The crystal structure of Atg3 shows a hammer-like conformation with an N-terminal head moiety and a Cterminal handle moiety [161]. The head moiety shares some similarities to canonical E2 enzymes, while the handle moiety, which is suggested to bind Atg7, is unique to Atg3.…”