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. 2013 Dec;22(12):1691-7.
doi: 10.1002/pro.2381.

Binding to E1 and E3 is mutually exclusive for the human autophagy E2 Atg3

Yu Qiu  1 , Kay Hofmann, Julie E Coats, Brenda A Schulman, Stephen E Kaiser
Affiliations

Binding to E1 and E3 is mutually exclusive for the human autophagy E2 Atg3

Yu Qiu et al. Protein Sci. 2013 Dec.

Abstract

Ubiquitin-like proteins (UBLs) are activated, transferred and conjugated by E1-E2-E3 enzyme cascades. E2 enzymes for canonical UBLs such as ubiquitin, SUMO, and NEDD8 typically use common surfaces to bind to E1 and E3 enzymes. Thus, canonical E2s are required to disengage from E1 prior to E3-mediated UBL ligation. However, E1, E2, and E3 enzymes in the autophagy pathway are structurally and functionally distinct from canonical enzymes, and it has not been possible to predict whether autophagy UBL cascades are organized according to the same principles. Here, we address this question for the pathway mediating lipidation of the human autophagy UBL, LC3. We utilized bioinformatic and experimental approaches to identify a distinctive region in the autophagy E2, Atg3, that binds to the autophagy E3, Atg12∼Atg5-Atg16. Short peptides corresponding to this Atg3 sequence inhibit LC3 lipidation in vitro. Notably, the E3-binding site on Atg3 overlaps with the binding site for the E1, Atg7. Accordingly, the E3 competes with Atg7 for binding to Atg3, implying that Atg3 likely cycles back and forth between binding to Atg7 for loading with the UBL LC3 and binding to E3 to promote LC3 lipidation. The results show that common organizational principles underlie canonical and noncanonical UBL transfer cascades, but are established through distinct structural features.

Keywords: Atg12∼Atg5; Atg3; Atg7; E1 enzyme; E1‐E2 binding; E2 enzyme; E2‐E3 binding; E3 enzyme; autophagy; ubiquitin‐like protein.

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Figures

Figure 1
Figure 1
LC3 conjugation pathway. (A) Atg8 family members such as human LC3 are activated by the E1 Atg7 and then transferred to the E2 enzyme, Atg3. Atg12∼Atg5-Atg16 acts as an E3 enzyme that catalyzes lipid conjugation of LC3 and other Atg8 protein family members. (B) Domain diagram representing the primary structure of Atg3 and highlighting the Atg3 Flexible Region. E2-N and E2-C are the N- and C-terminal parts of an E2 fold.
Figure 2
Figure 2
Atg7 and Atg12∼Atg5-Atg16L1 bind to an overlapping region in the Atg3 flexible region (FR). (A) Coomassie-stained SDS-PAGE gels of affinity pulldowns with purified recombinant GST-Atg7 and wild-type (wt) or deletion mutant versions of Atg3. Lanes showing input material are labeled “i” and lanes with affinity pulldowns are labeled “p.” (B) Affinity pulldown with purified recombinant GST-Atg12∼Atg5-his-MBP-Atg16L1 complex and wild-type or deletion mutant versions of Atg3.
Figure 3
Figure 3
Competition experiments. (A,B) 20 μM Atg7-Atg3 or Atg7-Atg3(Δ145–160) complex was incubated with increasing amounts of Atg12∼Atg5-Atg16L1 complex (0, 4, 10, 20 μM) as described under “Materials and Methods.” Atg3, Atg12∼Atg5-Atg16L1 complex, the Atg7-Atg3 complex, and the Atg3-Atg12∼Atg5-Atg16L1 complex were identified by their different migrations on a Coomassie-stained nondenaturing polyacrylamide gel and are indicated at the right. (C) Binding competition assay performed as for A and B showing titration of Atg3-Atg12∼Atg5-Atg16L1 with Atg7. (D) LC3 lipidation competition assays. Coomassie-stained urea SDS-PAGE showing in vitro LC3 lipidation in multiple turnover assays containing Atg7, Atg3, Atg12∼Atg5, and LC3. GST or GST-Atg3 peptides were added at a 10-fold molar excess over Atg3. (E) LC3 lipidation competition assays performed in the absence of E3 enzyme. Sypro-stained urea SDS-PAGE showing in vitro LC3 lipidation in multiple turnover assays containing Atg7, Atg3, and LC3. GST or GST-Atg3 peptides were added at a 10-fold molar excess over Atg3.
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References

    1. Geng J, Klionsky DJ. The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. Protein modifications: beyond the usual suspects review series. EMBO Rep. 2008;9:859–864. - PMC - PubMed
    1. Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol. 2009;10:458–467. - PubMed
    1. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27:107–132. - PubMed
    1. Devenish RJ, Klionsky DJ. Autophagy: mechanism and physiological relevance 'brewed' from yeast studies. Front Biosci (Schol Ed) 2012;4:1354–1363. - PMC - PubMed
    1. Mizushima N, Noda T, Yoshimori T, Tanaka Y, Ishii T, George MD, Klionsky DJ, Ohsumi M, Ohsumi Y. A protein conjugation system essential for autophagy. Nature. 1998;395:395–398. - PubMed

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