A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy

Abstract

Macroautophagy is an intracellular, vesicle-mediated mechanism for the sequestration and ultimate lysosomal degradation of cytoplasmic proteins, organelles and macromolecules. The macroautophagy process and many of the autophagy-specific (Atg) proteins are remarkably well conserved in higher eukaryotes. In yeast, the Atg1 kinase complex includes Atg1, Atg13, Atg17, and at least four other interacting proteins, some of which are phosphorylated in a TOR-dependent manner, placing the Atg1 signaling complex downstream of a major nutrient-sensing pathway. Atg1 orthologs, including mammalian unc-51-like kinase 1 (ULK1), have been identified in higher eukaryotes and have been functionally linked to autophagy. This suggests that other components of the Atg1 complex exist in higher eukaryotes. Recently, a putative human Atg13 ortholog, FLJ20698, was identified by gapped-BLAST analysis. We show here that FLJ20698 (Atg13) is a ULK1-interacting phosphoprotein that is essential for macroautophagy. Furthermore, we identify a novel, human Atg13-interacting protein, FLJ11773, which we have termed Atg101. Atg101 is essential for autophagy and interacts with ULK1 in an Atg13-dependent manner. Additionally, we present evidence that intracellular localization of the ULK1 complex is regulated by nutrient conditions. Finally, we demonstrate that Atg101 stabilizes the expression of Atg13 in the cell, suggesting that Atg101 contributes to Atg13 function by protecting Atg13 from proteasomal degradation. Therefore, the identification of the novel protein, Atg101, and the validation of Atg13 and Atg101 as ULK1-interacting proteins, suggests an Atg1 complex is involved in the induction of macroautophagy in mammalian cells.

Overview of macroautophagy regulation in mammalian cells

“…This differs from what is observed in yeast, where the inactivation of TOR increases the binding affinity of Atg1 to Atg13 and Atg17 [13]. Moreover, a novel mammalian Atg13-binding protein, Atg101, which is not conserved in Saccharomyces cerevisiae, has been identified by two teams simultaneously [21,22]. Atg101 is essential for autophagy; it localizes to the phagophore and stabilizes the expression of Atg13.…”

The machinery of macroautophagy

“…The mammalian ULK1/2 complex includes ULK1/2 (mammalian homologs of Atg1), ATG13 (a homolog of yeast Atg13), RB1CC1/FIP200 (a putative Atg17 homolog) and C12orf44/ATG101 (the latter component is not conserved in S. cerevisiae) [76][77][78][79][80]. ULK1/2 interact with ATG13, which directly binds RB1CC1 and mediates the latter's interaction with the ULKs [76,81].…”

Vesicular trafficking and autophagosome formation

“…The mAtg13 has recently been identified and was shown to be required for autophagy. 38,[41][42][43][44] It binds ULK1 and ULK2 at the C-terminal domain (CTD) independent of its phosphorylation state; however, mAtg13 was shown to be a substrate for ULK1 and ULK2. 38,[41][42][43] Interestingly, ULK1/2 and mAtg13 form a tight association with membranes and exhibit a partial colocalization.…”

“…42 Atg101, identified through its homology to a Atg1-binding protein in Drosophila, was shown to interact with the ULK1-Atg13-FIP200 complex in an Atg13 dependent manner. 44 The identification and characterization of potential ULK1/2 effectors will allow a better understanding of the function of these kinases in autophagosome formation. The two most promising avenues would be a further understanding of the membrane association of ULK1/2, mAtg13, and mTOR, how and where the kinases are activated, and how their activity regulates the initiation of autophagy and expansion of the IM.…”