Delineating the lipidated Atg8 structure for unveiling its hidden activity in autophagy

ABSTRACT

Atg8 has attracted attention as a central factor in autophagosome biogenesis for a long time. However, the molecular activities of Atg8 on the phagophore membranes as the physiologically functional lipidated form remain enigmatic. In our recent study, we unveiled the hidden physicochemical activity of lipidated Atg8 toward the membrane. Structural analysis revealed that lipidated Atg8 adopts a preferred orientation on the membrane, contacting the membrane using aromatic residues and at the same time exposing cargo binding pockets to the solvent, enabling this small protein to perturb and transform membranes while recognizing autophagic cargos. The membrane perturbation activity was shown to be essential for efficient autophagosome biogenesis, yet questions on the mechanistic roles of Atg8 remain open.

KEYWORDS:

• Atg8
• autophagosome
• autophagy
• GUV
• lipidation
• membrane perturbation
• NMR
• vacuolar morphology

Autophagosome biogenesis is an essential process in macroautophagy/autophagy that is mediated by ~20 autophagy-related (Atg) proteins in a membranous environment. Among them, the ubiquitin-like protein Atg8 is the sole protein that is abundant on the autophagosomal membranes. Atg8 undergoes lipidation that is mediated by ubiquitination-like conjugation systems: the C terminus of Atg8 is covalently attached to the headgroup of phosphatidylethanolamine (PE). As the lipidated form, Atg8–PE engages in at least two events in autophagy: autophagosome biogenesis and selective engulfment of cargos into phagophores. Previous studies have unveiled two molecular activities of Atg8, one is to recognize the Atg8-family interacting motif (AIM) in cargo receptors and another is to mediate tethering and hemi/full-fusion of membranes. However, it remains elusive how Atg8–PE on the membrane recognizes the AIM and how Atg8–PE contributes to autophagosome biogenesis using its membrane-related activities. The delayed understanding of these issues despite the tremendous amounts of studies on Atg8 is partly due to the lack of structural information on Atg8–PE in the membranous environment, the functional state of this protein.

Figure 1. Putative roles of membrane perturbation by Atg8–PE. Phe77 and Phe79 in Atg8 increase the area difference between outer and inner layers, Δa, in a lipidation-dependent manner. The membrane perturbation activity can affect membrane morphology. The activity of Atg8–PE may promote the formation of tubulovesicular structures containing Atg9 at the autophagosome formation site and/or the Atg2-mediated lipid transfer for autophagosome biogenesis [1]

In our recent study [1], we investigated the molecular activities of Saccharomyces cerevisiae Atg8–PE on the membrane structure by applying biophysical techniques using micropipettes to the in vitro reconstitution system of Atg8 lipidation. Non-spherical giant unilamellar vesicles (GUVs) were used as a model membrane. Local application of proteins via micropipette under confocal microscopy was helpful for monitoring flaccid, non-spherical GUVs in real-time. Upon the formation of Atg8–PE, prolate GUVs undergo drastic shape changes into spheres with out-budding. Importantly, membrane morphology is not changed by the chemical linkage of Atg8 with phospholipid, suggesting that the lipidation reaction by the Atg8 conjugation system is essential for Atg8–PE to cause membrane deformation. Moreover, the micropipette aspiration technique clarified that Atg8–PE expands the membrane area. Because Atg8–PE is produced only on the outer layer of GUVs, Atg8–PE interaction with the membrane expands the area of the outer layer, which then extends the area of the inner layer via hydrophobic interaction between acyl chains. Such an asymmetrical interaction can increase area difference between outer and inner layers, Δa, inducing morphological changes of GUVs. Together, the shape changes of non-spherical GUVs upon Atg8 lipidation shed light on the hidden physicochemical activity of Atg8–PE: increasing Δa in membranes.

Next, we used solution nuclear magnetic resonance (NMR) spectroscopy to examine Atg8–PE structure on the membrane. Atg8–PE with selective methyl-labeling was produced through enzyme reactions on nanodiscs that encapsulate lipid bilayer membranes. NMR analyses using the paramagnetic relaxation enhancement effect identified the residues in Atg8–PE in close proximity to the membrane while unlipidated Atg8 does not interact with the membrane. Based on the NMR data, we built the structural model of Atg8–PE on the nanodisc, which indicates that Atg8–PE adopts a preferred orientation on the membrane despite the anchoring to the membrane via the flexible C-terminal tail. The obtained model showed that Atg8–PE on the membrane exposes the AIM-binding pockets to the solvent, confirming its role in cargo recognition during selective autophagy.(Figure 1)

Based on the structure of Atg8–PE on the membrane, we then postulated that membrane-facing aromatic residues, Phe77 and Phe79, induce the aforementioned membrane deformation of GUVs. This hypothesis was validated by mutational analyses using Ala mutants for Phe77 or Phe79 in Atg8, which lose the activity to cause membrane-area expansion and to induce a drastic shape change of non-spherical GUVs. The physiological importance of membrane-facing aromatic residues in Atg8–PE was then examined in vivo. Fission yeast Atg8 regulates vacuolar morphology, maintaining a fragmented morphology of vacuoles under stress conditions. Mutational analyses on the aromatic residues in Atg8 showed that the vacuole is fragmented in the steady-state condition whereas it became an elongated tubular structure under stress conditions, illuminating the fact that the aromatic residues regulate the membrane morphology in vivo. We then studied the roles of the aromatic residues in autophagy. Budding yeast cells expressing Ala mutants for Phe77 or Phe79 in Atg8 exhibit fewer and smaller autophagosomes and autophagic bodies than those expressing wild-type Atg8. Moreover, mammalian cells expressing Ala mutants for the equivalent aromatic residues in LC3 or GABARAP, mammalian orthologs of yeast Atg8, show delayed clearance of SQSTM1/p62 bodies that accumulate inside the cell upon exposure to sodium arsenite. These data suggest that the membrane perturbation activity of Atg8 is important for efficient autophagosome biogenesis, and that this activity is evolutionarily conserved.

Our study revealed the structural basis for the physicochemical activity of Atg8–PE to perturb and transform membranes by increasing Δa, which is essential for efficient autophagosome biogenesis. However, it remains enigmatic when and where the membrane perturbation activity of Atg8–PE is effective during autophagy. Given the reduced number and smaller size of autophagosomes in the absence of the membrane perturbation activity of Atg8–PE, it may be related to membrane supply for nucleation and expansion steps during autophagosome biogenesis. One possible role is to promote formation of Atg9-resident tubulovesicular structures that are proposed to supply membranes for autophagosome biogenesis, and another is to facilitate the Atg2-mediated lipid transfer by reducing the energy barrier for extracting lipids from source membranes. Future study on the cooperative function of Atg8–PE with other Atg proteins in the membranous environment will provide further mechanistic insights into autophagosome biogenesis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported in part by JSPS KAKENHI grant nos. 18H03989 and 19H05707 (to N.N.N.); CREST, Japan Science and Technology Agency Grant number JPMJCR13M7 (to N.N.N.); JPMJCR20E3 (to N.N.N.); and grants from the Takeda Science Foundation (to N.N.N.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (to N.N.N.) and the Tokyo Biochemical Research foundation (to N.N.N.).