Bueno-Arribas, MirandaCruz-Cuevas, CeliaMonforte-Martinez, BeatrizNavas Hernández, María ÁngelesEscalante, RicardoVincent, Olivier2025-01-242025-01-242025Bueno-Arribas M, Cruz-Cuevas C, Monforte-Martinez B, Navas MA, Escalante R, Vincent O. The PKA signaling pathway regulates the association of the autophagy initiation complex with the lipidation machinery. J Mol Biol. 2025. 437(5):168954.0022-283610.1016/j.jmb.2025.168954https://hdl.handle.net/20.500.14352/116079A key step in autophagy is the conjugation by the E3-like Atg12-Atg5-Atg16 complex of the ubiquitin-like protein Atg8 to phosphatidylethanolamine on the autophagosomal membrane, a process known as lipida-tion. Previous work in yeast showed that recruitment of the E3-like complex to the preautophagosomal structure is mediated by the interaction of Atg16 with the phosphatidylinositol 3-phosphate-binding protein Atg21, and by the association of Atg12 with the scaffold protein of the Atg1 kinase complex, Atg17. Here, we conducted a reverse two-hybrid screen to identify residues in Atg17 and Atg12 critical for Atg17-Atg12 binding, and used these data to generate a docking model of Atg12-Atg5-Atg16 with the Atg17 complex. In this model, a conserved alpha-helix in the N-terminal region of Atg12 binds to the convex side of crescent-shaped Atg17 and appears to form a four-helix bundle with the three helices of Atg17, similar to that described for the binding of Atg31 to Atg17. We further showed that, in agreement with previous work, Atg17-Atg12 and Atg21-Atg16 binding act cooperatively to mediate the recruitment of the E3-like complex, although our results show that alternative mechanisms are involved in this process. Finally, we found that phosphorylation of Atg12 by PKA prevents its interaction with Atg17, thus adding a new reg-ulatory layer in the control of autophagy by the PKA signaling pathway.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal articlehttps://doi.org/10.1016/j.jmb.2025.168954https://www.sciencedirect.com/science/article/pii/S0022283625000208?via%3Dihubopen access577.2AutophagyATG17ATG12PKAReverse two-hybridCiencias24 Ciencias de la Vida