The insufficiency of ATG4A in macroautophagy.

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.

Maturation and Clearance of Autophagosomes in Neurons Depends on a Specific Cysteine Protease Isoform, ATG-4.2.

In neurons, defects in autophagosome clearance have been associated with neurodegenerative disease. Yet, the mechanisms that coordinate trafficking and clearance of synaptic autophagosomes are poorly understood. Here, we use genetic screens and in vivo imaging in single neurons of C. elegans to identify mechanisms necessary for clearance of synaptic autophagosomes. We observed that autophagy at the synapse can be modulated in vivo by the state of neuronal activity, that autophagosomes undergo UNC-16/JIP3-mediated retrograde transport, and that autophagosomes containing synaptic material mature in the cell body. Through forward genetic screens, we then determined that autophagosome maturation in the cell body depends on the protease ATG-4.2, but not the related ATG-4.1, and that ATG-4.2 can cleave LGG-1/Atg8/GABARAP from membranes. Our studies revealed that ATG-4.2 is specifically necessary for the maturation and clearance of autophagosomes and that defects in transport and ATG-4.2-mediated maturation genetically interact to enhance abnormal accumulation of autophagosomes in neurons.

Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes.

Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine 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 phosphatidylethanolamine at the target membrane. Here we have purified the full-length human ATG12-5-ATG16L1 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 membrane-binding amphipathic helix is required for LC3B lipidation under all conditions tested. By 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. This C-terminal membrane-binding region is present only in the ß-isoform of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish between different LC3B lipidation mechanisms under different cellular conditions.

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.

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 Legionella Anti-autophagy Effector RavZ Targets the Autophagosome via PI3P- and Curvature-Sensing Motifs.

Autophagy is a conserved membrane transport pathway used to destroy pathogenic microbes that access the cytosol of cells. The intracellular pathogen Legionella pneumophila interferes with autophagy by delivering an effector protein, RavZ, into the host cytosol. RavZ acts by cleaving membrane-conjugated Atg8/LC3 proteins from pre-autophagosomal structures. Its remarkable efficiency allows minute quantities of RavZ to block autophagy throughout the cell. To understand how RavZ targets pre-autophagosomes and specifically acts only on membrane-associated Atg8 proteins, we elucidated its structure. Revealed is a catalytic domain related in fold to Ulp family deubiquitinase-like enzymes and a C-terminal PI3P-binding module. RavZ targets the autophagosome via the PI3P-binding module and a catalytic domain helix, and it preferentially binds high-curvature membranes, intimating localization to highly curved domains in autophagosome intermediate membranes. RavZ-membrane interactions enhance substrate affinity, providing a mechanism for interfacial activation that also may be used by host autophagy proteins engaging only lipidated Atg8 proteins.

The interaction of polyglutamine peptides with lipid membranes is regulated by flanking sequences associated with huntingtin.

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.

A stripe-to-droplet transition driven by conformational transitions in a binary lipid-lipopolymer mixture at the air-water interface.

We report the observation of an unusual stripe-droplet transition in precompressed Langmuir monolayers consisting of mixtures of poly(ethylene) glycol (PEG) amphiphiles and phospholipids. This highly reproducible and fully reversible transition occurs at approximately zero surface pressure during expansion (or compression) of the monolayer following initial compression into a two-dimensional solid phase. It is characterized by spontaneous emergence of an extended, disordered stripe-like morphology from an optically homogeneous phase during gradual expansion. These stripe patterns appear as a transient feature and continuously progress, involving gradual coarsening and ultimate transformation into a droplet morphology upon further expansion. Furthermore, varying relative concentrations of the two amphiphiles and utilizing amphiphiles with considerably longer ethylene glycol headgroups reveal that this pattern evolution occurs in narrow concentration regimes, values of which depend on ethylene oxide headgroup size. These morphological transitions are reminiscent of those seen during a passage through a critical point by variations in thermodynamic parameters (e.g., temperature or pressure) as well as those involving spinodal decomposition. While the precise mechanism cannot be ascertained using present experiments alone, our observations can be reconciled in terms of modulations in competing interactions prompted by the pancake-mushroom-brush conformational transitions of the ethylene glycol headgroup. This in turn suggests that the conformational degree of freedom represents an independent order parameter, or a switch, which can induce large-scale structural reorganization in amphiphilic monolayers. Because molecular conformational changes are pervasive in biological membranes, we speculate that such conformational transition-induced pattern evolution might provide a physical mechanism by which membrane processes are amplified.