Optineurin promotes autophagosome formation by recruiting the autophagy-related Atg12-5-16L1 complex to phagophores containing the Wipi2 protein

Megha Bansal, Shivranjani C. Moharir, S. Purnima Sailasree, Kapil Sirohi, Cherukuri Sudhakar, D. Partha Sarathi, B. Jyothi Lakshmi, Mario Buono, Satish Kumar, Ghanshyam Swarup
2017 Journal of Biological Chemistry  
Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for
more » ... ich is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores. Macroautophagy, called autophagy here, is an evolutionarily conserved cellular degradative process, which represents path-ways leading to lysosomal degradation of cytoplasmic constituents, from macromolecules to organelles (1, 2). It is also involved in development, immunity, and clearance of invading microorganisms and its impairment contributes to several diseases (1, 3). Generally autophagy occurs at a basal level, and it is enhanced by several types of stresses, such as starvation, damaged organelles, abnormal proteins, and invading pathogens (4). Autophagy is initiated by signaling events that lead to formation of a cup-shaped double membrane structure known as phagophore (isolation membrane), which matures into autophagosome (1, 5). Many autophagy-related (Atg) proteins in yeast and mammals form the core autophagy machinery (1, 6). Formation of phagophore is initiated by activation and translocation of ULK1 protein kinase from cytosol to the endoplasmic reticulum (7). This step is regulated by mammalian target of rapamycin (mTOR) 3 kinase, a cell growth regulator, which inhibits autophagy when nutrients are available in the cell, by phosphorylating ULK1. ULK1 is positively regulated by AMP-activated protein kinase (AMPK), an energy sensor (8). Activated ULK1 complex initiates autophagy by phosphorylating components of type III phosphatidylinositol 3-kinase complex, which contains VPS34 and Beclin1. Phosphorylated Beclin1 promotes generation of phosphatidylinositol 3-phosphate (PI3P) by VPS34, which recruits effectors such as DFCP1 and the WIPI (WD-repeat phosphoinositide interacting) family of proteins (9, 10). These proteins then recruit ATG12-5-16L1 complex and ATG3-LC3 conjugate to the phagophore (11). ATG5, which is conjugated to ATG12, interacts with ATG16L1 (12). ATG12-5-16L1 complex functions as E3 ligase in a ubiquitination-like reaction that conjugates LC3-I with phosphatidylethanolamine to produce LC3-II, which is membraneassociated. The LC3 family of proteins is needed for several functions such as expansion and closure of phagophore to form autophagosome, and recruitment of cargo (13, 14) . During formation of autophagosomes, the cargo that needs to be degraded is recruited to autophagosomes by specialized
doi:10.1074/jbc.m117.801944 pmid:29133525 fatcat:b3aol6o3ezhg5hu5ch2evreime