Mechanism of cargo-directed Atg8 conjugation during selective autophagy

Abstract

Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.DOI: http://dx.doi.org/10.7554/eLife.18544.001

p62 filaments capture and present ubiquitinated cargos for autophagy

“…During selective autophagy, the cargos themselves drive the local assembly of autophagosomes and keep the membrane close to the cargo (Sawa‐Makarska et al , ; Lazarou et al , ; Bertipaglia et al , ; Fracchiolla et al , ; Zaffagnini & Martens, ). Here, we show that within the p62 clusters in vitro, the p62 filaments are largely immobile.…”

Intrinsic lipid binding activity of ATG 16L1 supports efficient membrane anchoring and autophagy

“…Further analyses of the CCD domain region indicated the presence of a hydrophobic region and positively charged residues that could mediate direct lipid binding of ATG16L1 to membranes ( Fig 3A). To address this possibility, we used a microscopy-based technique to test the recruitment of rhodaminelabelled small unilamellar vesicles (SUVs) to beads coated with ATG16L1-GFP (Fracchiolla et al, 2016), a sensitive approach to detect protein-lipid binding activities. As seen in Fig 3B and C, there was no significant recruitment of liposomes to ATG16L1-GFP-bound beads when using liposome preparations that contained phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS) suggesting an inability of ATG16L1 to bind these phospholipids.…”

“…Beads were washed four times with liposome binding buffer. For microscopy-based protein-liposome interaction assay (Fracchiolla et al, 2016), 1 ll of StrepTactin beads covered with ATG16L1 was incubated with 15 ll of liposomes (1 mg/ml) prepared by extrusion (100 nm) and contained the following composition 39.5% DOPC, 35% DOPS, 20% DOPE, 5% phosphoinositides (as indicated in the figure) and 0.5% rhodamine-phosphoethanolamine (L1392, Thermo scientific). The binding reaction was incubated for 15 min at room temperature.…”

A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy

“…Macroautophagy (hereafter autophagy) is a conserved intracellular degradation process that ensures cellular homeostasis by the degradation of harmful material such as damaged organelles, misfolded proteins and bacterial pathogens. It also ensures survival of cells during starvation (Anding and Baehrecke, 2017;Gomes and Dikic, 2014;Kirkin and Rogov, 2019;Wen and Klionsky, 2016;Zaffagnini and Martens, 2016). These functions are achieved by the sequestration of the cargo material within double membrane vesicles, the autophagosomes, which form de novo around the cargo.…”

“…The E3 complex subsequently acts to promote the attachment of LC3 proteins to the membrane lipid phosphatidylethanolamine (PE) in a manner analogous to the action of E3-ligases in ubiquitin conjugation reactions (Hanada et al, 2007). The attachment of LC3 to PE, referred to as lipidation, requires the E1-like ATG7 as well as the E2-like ATG3 enzymes, and mediates efficient phagophore elongation and serves to recruit cargo material into autophagosomes (Ichimura et al, 2000;Zaffagnini and Martens, 2016). The E3 complex itself is the product of a ubiquitin-like conjugation machinery wherein the ATG7 and ATG10 proteins conjugate the ubiquitin-like ATG12 to a lysine residue in ATG5 (Mizushima et al, 1998).…”