Shuttling of macromolecules between different cellular compartments helps regulate the timing and extent of different cellular activities. Here, we report that LC3, a key initiator of autophagy that cycles between the nucleus and cytoplasm, becomes selectively activated in the nucleus during starvation through deacetylation by the nuclear deacetylase Sirt1. Deacetylation of LC3 at K49 and K51 by Sirt1 allows LC3 to interact with the nuclear protein DOR and return to the cytoplasm with DOR, where it is able to bind Atg7 and other autophagy factors and undergo phosphatidylethanolamine conjugation to preautophagic membranes. The association of deacetylated LC3 with autophagic factors shifts LC3's distribution from the nucleus toward the cytoplasm. Thus, an acetylation-deacetylation cycle ensures that LC3 effectively redistributes in an activated form from nucleus to cytoplasm, where it plays a central role in autophagy to enable the cell to cope with the lack of external nutrients.

Acetylation is increasingly recognized as one of the major post-translational mechanisms for the regulation of multiple cellular functions in mammalian cells. Acetyltransferase p300, which acetylates histone and non-histone proteins, has been intensively studied in its role in cell growth and metabolism. However, the mechanism underlying the activation of p300 in cells remains largely unknown. Here, we identify the homeostatic sensor mTORC1 as a direct activator of p300. Activated mTORC1 interacts with p300 and phosphorylates p300 at 4 serine residues in the C-terminal domain. Mechanistically, phosphorylation of p300 by mTORC1 prevents the catalytic HAT domain from binding to the RING domain, thereby eliminating intra-molecular inhibition. Functionally, mTORC1-dependent phosphorylation of p300 suppresses cell-starvation-induced autophagy and activates cell lipogenesis. These results uncover p300 as a direct target of mTORC1 and suggest that the mTORC1-p300 pathway plays a pivotal role in cell metabolism by coordinately controlling cell anabolism and catabolism.

Autophagy receptor p62/SQSTM1 promotes the assembly and removal of ubiquitylated proteins by forming p62 bodies and mediating their encapsulation in autophagosomes. Here we show that under nutrient-deficient conditions, cellular p62 specifically undergoes acetylation, which is required for the formation and subsequent autophagic clearance of p62 bodies. We identify K420 and K435 in the UBA domain as the main acetylation sites, and TIP60 and HDAC6 as the acetyltransferase and deacetylase. Mechanically, acetylation at both K420 and K435 sites enhances p62 binding to ubiquitin by disrupting UBA dimerization, while K435 acetylation also directly increases the UBA-ubiquitin affinity. Furthermore, we show that acetylation of p62 facilitates polyubiquitin chain-induced p62 phase separation. Our results suggest an essential role of p62 acetylation in the selective degradation of ubiquitylated proteins in cells under nutrient stress, by specifically regulating the assembly of p62 bodies.

Highlights d HUWE1 mediates the ubiquitination and degradation of WIPI2 d mTORC1 promotes WIPI2 degradation by phosphorylating WIPI2 d Phosphorylation at Ser395 of WIPI2 enhances HUWE1 interaction d WIPI2 level determines autophagy flux and lipid clearance

Accumulating evidence highlights the role of histone acetyltransferase GCN5 in the regulation of cell metabolism in metazoans.Here, we report that GCN5 is a negative regulator of autophagy, a lysosome-dependent catabolic mechanism. In animal cells and Drosophila, GCN5 inhibits the biogenesis of autophagosomes and lysosomes by targeting TFEB, the master transcription factor for autophagy-and lysosome-related gene expression. We show that GCN5 is a specific TFEB acetyltransferase, and acetylation by GCN5 results in the decrease in TFEB transcriptional activity. Induction of autophagy inactivates GCN5, accompanied by reduced TFEB acetylation and increased lysosome formation. We further demonstrate that acetylation at K274 and K279 disrupts the dimerization of TFEB and the binding of TFEB to its target gene promoters. In a Tau-based neurodegenerative Drosophila model, deletion of dGcn5 improves the clearance of Tau protein aggregates and ameliorates the neurodegenerative phenotypes. Together, our results reveal GCN5 as a novel conserved TFEB regulator, and the regulatory mechanisms may be involved in autophagy-and lysosome-related physiological and pathological processes. ª 2019 The Authors. Published under the terms of the CC BY NC ND 4.0 license EMBO reports 21: e48335 | 2020 H LC3-II formation in GFP-GCN5-overexpressing HeLa cells. I GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were cultured with GCN5 siRNA with or without CQ. J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP-GCN5 and addition of CQ. KRepresentative images of mCherry-Atg8a (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD) using the pan-fat body driver (cg-GAL4). Drosophila (cg-GAL4/+) was used as the control (graph represents data from three independent experiments with ≥ 30 cells per condition; mean AE SEM; *P < 0.05, ***P < 0.001, Student's t-test; Scale bars, 10 lm).