Local membrane source gathering by p62 body drives autophagosome formation.

Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.

Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation.

Misfolded proteins can be degraded by selective autophagy. The prevailing view is that ubiquitin-tagged misfolded proteins are assembled into aggregates by the scaffold protein p62, and the aggregates are then engulfed and degraded by autophagosomes. Here we report that p62 forms droplets in vivo which have liquid-like properties such as high sphericity, the ability to undergo fusion, and recovery after photobleaching. Recombinant p62 does not undergo phase separation in vitro; however, adding a K63 polyubiquitin chain to p62 induces p62 phase separation, which results in enrichment of high-molecular weight ubiquitin signals in p62 droplets. Mixing recombinant p62 with cytosol from p62-/- cells also results in p62 phase separation in a polyubiquitination-dependent manner. Mechanistically, p62 phase separation is dependent on p62 polymerization, the interaction between p62 and ubiquitin, and the valence of the polyubiquitin chain. Moreover, p62 phase separation can be regulated by post-translational modifications such as phosphorylation. Finally, we demonstrate that disease-associated mutations in p62 can affect phase separation. We propose that polyubiquitin chain-induced p62 phase separation drives autophagic cargo concentration and segregation.

Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation.

PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation.

Function and molecular mechanism of acetylation in autophagy regulation.

Protein acetylation emerged as a key regulatory mechanism for many cellular processes. We used genetic analysis of Saccharomyces cerevisiae to identify Esa1 as a histone acetyltransferase required for autophagy. We further identified the autophagy signaling component Atg3 as a substrate for Esa1. Specifically, acetylation of K19 and K48 of Atg3 regulated autophagy by controlling Atg3 and Atg8 interaction and lipidation of Atg8. Starvation induced transient K19-K48 acetylation through spatial and temporal regulation of the localization of acetylase Esa1 and the deacetylase Rpd3 on pre-autophagosomal structures (PASs) and their interaction with Atg3. Attenuation of K19-K48 acetylation was associated with attenuation of autophagy. Increased K19-K48 acetylation after deletion of the deacetylase Rpd3 caused increased autophagy. Thus, protein acetylation contributes to control of autophagy.