The human autophagy-initiating complexes ULK1C and PI3KC3-C1

The unc-51-like kinase complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) are the key regulators of macroautophagy initiation.Understanding the assembly and coordination of these two complexes is essential for deciphering their cellular regulation and targeting them for therapeutic enhancement.This review highlights recent advances in our understanding of the structural organization and activation mechanisms of ULK1C and PI3KC3-C1 at the molecular level and discusses their roles within the protein interaction network governing autophagy initiation.J o u r n a l P r e -p r o o f ATG12-ATG5-ATG16L1 has a ubiquitin E3-like activity in promoting ATG8 family protein conjugation to the membrane lipid phosphatidylethanolamine (PE) and is often referred to as the "E3" in this context (Figure 1B).Formation of the E3 complex is dependent on a ATG12-ATG5 conjugation reaction catalyzed by ATG7 (E1-like) and ATG10 (E2-like) proteins.Meanwhile, ATG2A establishes contact sites between phagophores and the membrane sources such as ER, facilitating lipid transfer to the expanding phagophore (21-23) (Figure 1B).The final autophagy core complex, ATG9A, act as a scramblase within the phagophore membrane, balancing lipid distribution across the outer and inner leaflets created by ATG2 (24-28).Vesicles containing ATG9A, known as ATG9 vesicles, are proposed to serve as both the initial seeds and lipid sources for the phagophore formation (10, 29) (Figure 1A).In this review, we highlight recent advancement in understanding the first two core complexes, ULK1C and PI3KC3-C1.The latest molecular studies on the structure, assembly, coordination, and activation of these complexes shed light on the mechanism underlying phagophore formation.These findings offer a detailed view of their regulation in atomistic detail and suggest concepts for their therapeutic targeting in the future. Overview of the ULK1CThe ULK1C is composed of four subunits: the serine/threonine protein kinase ULK1, the scaffold protein focal adhesion kinase family interacting protein of 200 kDa (FIP200), and the regulatory proteins autophagy-related protein 13 (ATG13) and 101 (ATG101) (Figure 2A) (30)(31)(32)(33)(34). ULK1 is the catalytic subunit of the complex and comprises an N-terminal kinase domain (KD, residues 1-278), a long serineproline-rich region (279-827), and a C-terminal tandem microtubule-interacting and transport (MIT) domain (828-1050), also sometimes referred to as the early autophagy tethering/targeting (EAT) domain ( 35).The N-terminal domain of ULK1 adopts a typical kinase structure but features a non-conserved regulatory loop, possessing an autophosphorylation site at Thr180 which is crucial for the autophagy activity (36).At the other end of ULK1, the C-terminal MIT domain is responsible for interacting with other two ULK1C subunits FIP200 and ATG13 (37,38).FIP200 consists of an N-terminal scaffold domain (NTD) (1-640) which contains within it an ubiquitin-like domain (ULD) (1-80), an intrinsically disordered region (IDR, residues 641-790), a long coiled-coil (CC) domain (791-1497), and a C-terminal Claw domain .FIP200 forms a long, flexible homodimer that plays a central role in complex formation and interacts with various autophagy-related proteins (39).The remaining two subunits, ATG13 and ATG101, are primarily Hop1/Rev7/Mad2 (HORMA) proteins (40, 41).The N-terminal region of