P62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.

Inter-organelle membrane contact sites (MCSs) are classically defined as areas of close proximity between heterologous membranes and established by specific proteins (termed tethers). The interest on MCSs has rapidly increased in the last years, since MCSs play a crucial role in the transfer of cellular components between different organelles and have been involved in important cellular functions such as apoptosis, organelle division and biogenesis, and cell growth. Recently, an unprecedented depth and breadth in insights into the details of MCSs have been uncovered. On one hand, extensive MCSs (organelles interactome) are revealed by comprehensive analysis of organelle network with high temporal-spatial resolution at the system level. On the other hand, more and more tethers involving in MCSs are identified and further works are focusing on addressing the role of these tethers in regulating the function of MCSs at the molecular level. These enormous progresses largely depend on the powerful approaches, including several different types of microscopies and various biochemical techniques. These approaches have greatly accelerated recent advances in MCSs at the system and molecular level. In this review, we summarize the current and emerging approaches for studying MCSs, such as various microscopies, proximity-driven fluorescent signal generation and proximity-dependent biotinylation. In addition, we highlight the advantages and disadvantages of the techniques to provide a general guidance for the study of MCSs.

Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12-ATG5 conjugate to form an ATG12-ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1^C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.