FIP200 organizes the autophagy machinery at p62-ubiquitin condensates beyond activation of the ULK1 kinase

Abstract

<jats:title>Abstract</jats:title><jats:p>Macroautophagy is a conserved degradation pathway, which mediates cellular homeostasis by the delivery of harmful substances into lysosomes. This is achieved by the sequestration of these substances referred to as cargo within double membrane vesicles, the autophagosomes, which form <jats:italic>de novo</jats:italic>. Among the many cargoes that are targeted by autophagy are condensates containing p62 and ubiquitinated proteins. p62 recruits the FIP200 protein to initiate autophagosome formation at the condensates. How FIP200 in turn organizes the autophagy machinery is unclear. Here we show that FIP200 is dispensable for the recruitment of the upstream autophagy machinery to the condensates, but it is necessary for phosphatidylinositol 3-phosphate formation and WIPI2 recruitment. We further find that FIP200 is required for the activation of the ULK1 kinase. Surprisingly, ULK1 kinase activity is not strictly required for autophagosome formation at p62 condensates. Super-resolution microscopy of p62 condensates revealed that FIP200 surrounds the condensates where it spatially organizes ATG13 and ATG9A for productive autophagosome formation. Our data provide a mechanistic insight into how FIP200 orchestrates autophagosome initiation at the cargo.</jats:p>

Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy

“…We found, however, that the KD version of the ULK1 complex was as effective in promoting LC3 lipidation as wild type. This result is consistent with recent studies that found ULK1 kinase activity to be dispensable for PI3KC3-C1 activation induced by starvation (60) and autophagosome formation at p62 condensates (61).…”

The selective autophagy adaptor p62/SQSTM1 forms phase condensates regulated by HSP27 that facilitate the clearance of damaged lysosomes via lysophagy

“…FIP200 recruitment results in the local generation of phosphatidylinositol 3-phosphate, which in turn induces recruitment of WIPI2. 37 , 62 Therefore, we assayed whether p62 is required for Halo-WIPI2b puncta formation following 2 h of 750 μM LLOMe. We observed that p62 knockdown resulted in significantly fewer WIPI2b puncta as compared with control siRNA ( Figures 4F and 4G ), whereas co-expression of siRNA-resistant WT p62 construct rescued formation of WIPI2b puncta to control levels.…”

The crucial role of the regulatory mechanism of the Atg1/ULK1 complex in fungi

“…And the N-terminal domain (NTD) of the FIP200 forms a C-type scaffold, providing a hub for the assembly of the ULK1 complex ( Shi et al, 2020b ). Besides, FIP200 organizes autophagic mechanisms at p62-ubiquitin condensates beyond to ULK1 kinase activation ( Turco et al, 2020 ). And FIP200 coiled-coil and autophagic adapter NDP52 allosterically activate ULK1 complex membrane recruitment ( Shi et al, 2020a ).…”