No abstract

Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine (PE) in the double-membrane phagophore is a key event in the early phase of macroautophagy, but can also occur on single membrane structures. In both cases this involves transfer of LC3/GABARAP from ATG3 to PE at the target membrane. Here we have purified the full-length human ATG12-5-16L1 complex and show its essential role in LC3B/GABARAP lipidation in vitro. We have identified two functionally distinct membrane-binding regions in ATG16L1. An N-terminal membranebinding amphipathic helix is required for LC3B lipidation under all conditions tested. In contrast, the C-terminal membrane-binding region is dispensable for canonical autophagy, but essential for VPS34-independent LC3B-lipidation at perturbed endosomes. We further show that the ATG16L1 C-terminus can compensate for WIPI2 depletion to sustain lipidation during starvation. Remarkably, the C-terminal membrane-binding region comprises the β-isoform-specific sequence of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish LC3B-lipidation under different cellular conditions.

Autophagosome biogenesis involves de novo formation of a membrane that elongates to sequester cytoplasmic cargo and closes to form a double-membrane vesicle (an autophagosome). This process has remained enigmatic since its initial discovery >50 yr ago, but our understanding of the mechanisms involved in autophagosome biogenesis has increased substantially during the last 20 yr. Several key questions do remain open, however, including, What determines the site of autophagosome nucleation? What is the origin and lipid composition of the autophagosome membrane? How is cargo sequestration regulated under nonselective and selective types of autophagy? This review provides key insight into the core molecular mechanisms underlying autophagosome biogenesis, with a specific emphasis on membrane modeling events, and highlights recent conceptual advances in the field.

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.

The clearance of damaged or dysfunctional mitochondria by selective autophagy (mitophagy) is important for cellular homeostasis and prevention of disease. Our understanding of the mitochondrial signals that trigger their recognition and targeting by mitophagy is limited. Here we show that the mitochondrial matrix proteins NIPSNAP1 (4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1) and NIPSNAP2 accumulate on the mitochondria surface upon mitochondrial depolarization. There they recruit proteins involved in selective autophagy, including autophagy receptors and ATG8 proteins, thereby functioning as an "eat-me signal" for mitophagy. NIPSNAP1 and NIPSNAP2 have a redundant function in mitophagy and are predominantly expressed in different tissues, with NIPSNAP1 being the most abundant in the brain. Zebrafish lacking a functional Nipsnap1 display reduced mitophagy in the brain and parkinsonian phenotypes, including loss of tyrosine hydroxylase (Th1) positive dopaminergic (DA) neurons, reduced motor activity and increased oxidative stress.