ATG13: just a companion, or an executor of the autophagic program?

Abstract

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play.

Keywords: ATG101; ATG13; RB1CC1; ULK1; autophagy.

Figure 1. Schematic representation of yeast and vertebrate Atg13/ATG13 proteins. (A) Yeast Atg13 contains an N-terminal HORMA domain and a central Atg1-Atg17-binding region. Jao et al. reported that the HORMA domain is required for the recruitment of Atg14. Within this domain, they identified R120, R213, L216, and S217 (amino acid positions of the S. cerevisiae protein; originally the crystal structure of L. thermotolerans was solved with the corresponding amino acid positions R118, R205, L208 and S209) to be involved in binding of a SO₄^2--ion from the crystallization medium, and they hypothesized that the 2 arginine residues might comprise a biological phosphate sensor. Kamada et al. mapped the Atg1-binding region to amino acids 432–520 within yeast Atg13. In 2012, Kraft et al. reported that mutation of F468 and V469 to alanine is sufficient to abolish Atg1 binding. The Atg17-binding site was mapped to amino acids 280–520 by Cheong et al. In 2010, Kamada et al. reported the identification and/or prediction of 8 TORC1-sites (S348, S437, S438, S496, S535, S541, S646, and S649). Residues S344, S437 and S581 were suggested as PKA-sites. (B) The human ATG13 isoforms 1, 2 and 3 are depicted. ATG13 directly interacts with both RB1CC1 and ULK1/2 via its C-terminal region. The binding site has been roughly mapped to residues 384–517 of isoform 1 by Jung et al. Our group was able to narrow down the RB1CC1-binding site in ATG13 to residues 348–373 of the full-length avian ATG13 protein, which corresponds to the human isoform 2. Terje Johansen’s group reported the existence of a LIR motif within the C-terminal part (amino acids 443–447 in isoform 1). Notably, isoform 3 lacks this LIR motif and ULK1-binding capacity, but not the RB1CC1-binding site identified by our group. Mercer et al. mapped the ATG101-binding region to amino acids 112–220, and recently Karanasios et al. reported that ATG13 contains an N-terminal membrane binding site with specificity for the acidic phospholipids phosphatidic acid (PA), PtdIns3P and PtdIns4P. Apparently, a conserved cluster of 1 arginine (R10) and 3 lysine residues (K11, K15 and K18) mediates this lipid binding capacity. Our group was able to identify 5 ULK1-dependent phospho-sites in vitro (corresponding to S48, T170, T331, T428 and T478 in human isoform 2), but their in vivo relevance awaits further clarification. Next to these sites, only 2 additional phospho-acceptor sites have been reported so far (corresponding to S318 and S324 in human isoform 2).^-

Figure 2. Initiation of autophagosome generation in yeast and vertebrates. (A) In yeast, autophagosomes originate from a single location near the vacuole, the so-called phagophore assembly site (PAS). The pre-assembled Atg17-Atg31-Atg29 complex resides at the PAS and functions as a platform for further downstream factors.^- In the initial stage of autophagosome generation, the serine/threonine kinase Atg1 is recruited to the PAS via the adaptor protein Atg13, which is largely dephosphorylated upon autophagy induction and subsequently interacts both with Atg1 and Atg17.^, The strong enhancement of Atg1 kinase activity observed during autophagy induction requires both Atg13 and Atg17 and is essential for further progression of autophagosome generation. The N-terminal HORMA domain of Atg13 is crucial for the subsequent recruitment of the Atg14-containing class III PtdIns3K complex I. The locally restricted production of the membrane lipid PtdIns3P (yellow dots) by PtdIns3K directs additional factors such as the Atg2-Atg18 complex to the PAS and leads to further expansion of the phagophore (PG). (B) Although the origin of autophagosomes is more ambiguous in vertebrates, the endoplasmic reticulum (ER) has been proposed as a major site for autophagosome generation. In possible contrast to yeast, the interactions between mammalian ATG13 and the respective counterparts of Atg1 (ULK1) and Atg17 (RB1CC1) are constitutive and all 3 proteins are part of a large complex that is recruited to the equivalent of the PAS upon autophagy induction. The complex additionally includes ATG101, which protects ATG13 from proteasomal degradation and has no corresponding counterpart in yeast.^, Upon autophagy induction, the cytosolic ULK1-ATG13-RB1CC1-ATG101 complex redistributes to the ER. There, the class III PtdIns3K, which includes ATG14 and BECN1, is activated, leading to the production of PtdIns3P and the nucleation of membrane compartments termed omegasomes. As in yeast, the locally restricted generation of PtdIns3P is a crucial event that recruits further downstream effectors such as ZFYVE1/DFCP1 or members of the WIPI family to the site of autophagosome generation.^, The subsequent lipidation of ubiquitin-like molecules of the ATG8 family, such as the microtubule-associated protein 1 light chain 3 (LC3), with phosphatidylethanolamine (green dots) at the omegasome finally leads to the formation and elongation of the phagophore. For reasons of simplicity, ATG9-containing compartments and the Atg12/ATG12–Atg5/ATG5-Atg16/ATG16L1 complex are omitted in (A and B).