Autophagy is a degradation process of proteins in cells during a state of nutrient starvation. It is a basic and primary function found in almost all eukaryotes, from yeasts to humans. Since abnormalities in autophagy cause neurodegenerative diseases and cancer, understanding its mechanism is also essential for the development of treatments and prevention of diseases. However, the mechanism and nature of the structures responsible for autophagy in a cell have long been unknown. Little is known about the mechanism of autophagy, in particular, how autophagosomes, which are pouch-like lipid membranes holding degradation targets, expand.

To address this issue, Noda's group in CREST has elucidated that the structure responsible for autophagy is a liquid droplet^*1formed by "liquid-liquid phase separation^*2" of the Atg proteins (Fig. 1). This is a new discovery that shows that the phenomenon of liquid-liquid phase separation directly regulates autophagy. Noda et al. also found that the membrane protein Atg9 acts in the expansion of autophagosomes.

New drugs that regulate autophagy are expected to be developed in the future.

In autophagy, when the cell is nutrient-starved, a pouch-like lipid membrane called an autophagosome is created, and it holds degradation targets. They are then transported to a lysosome where degradation takes place. It was known that about 20 different Atg proteins were involved in the production of autophagosomes. It was also known that the Atg proteins aggregate to form a structure called PAS^*3. However, the mechanism of how Atg proteins aggregate and the nature of the PAS formed had not been well understood.

To address this issue, Noda's group used fluorescence microscopy to examine Atg13, a type of Atg protein, in detail. They further reconstructed PAS in vitro to study its behavior. They also analyzed PAS using a high-speed atomic force microscope^*4.

The results showed that PAS is a droplet formed by the liquid-liquid phase separation of Atg13 along with other Atg proteins, and that this droplet concentrates on the vacuolar membrane to form PAS, forming autophagosomes.

*3 PAS
PAS stands for "pre-autophagosomal structure", Atg proteins aggregate in a single location near the vacuole during nutrient starvation in yeast. PAS refers to this aggregated structure. Autophagosomes are presumably formed with PAS as a starting point.

*4 High-speed atomic force microscope
An atomic force microscope (AFM) is a microscope that visualizes the shape of molecules based on the atomic forces acting between the tip and the sample. Among them, a high-speed AFM is an instrument that can observe biomolecules such as proteins moving in solution with nanometer-scale spatial resolution and sub-second-scale time resolution.

The most important feature of autophagy is that it creates new autophagosomes. The process of forming autophagosomes determines the degradation targets. Approximately 20 types of Atg proteins are involved in the formation of autophagosomes, most of which have been analyzed for structure and function. Noda's group previously discovered that Atg2, a lipid transfer protein, carries phospholipids from endoplasmic reticula to form autophagosomes. However, how the lipid membrane of the autophagosome expands using the transported phospholipids was still completely unknown (Fig. 2).

They focused on Atg9, the only membrane protein whose function was unknown among the Atg proteins involved in autophagy. Using Atg9 extracted from yeast, they carefully examined its function in vitro. As a result, they discovered that Atg9 works to move the lipids that constitute the membrane of autophagosomes from one layer to the other of the "lipid bilayer" forming a liposome. The three-dimensional structure of yeast Atg9 examined by cryo-electron microscopy revealed that Atg9 has pores connecting the two layers of lipid bilayer (Fig. 3).

These findings indicate that Atg9 causes the expansion of autophagosome membranes. This means that Atg9 cooperates with Atg2 in the formation of autophagosome membranes (Fig. 4). This discovery is a new mechanism that has not previously been predicted, and is a breakthrough that will change the direction of basic research on autophagy.

The discovery of the direct regulation of autophagy by liquid-liquid phase separation is an achievement that once again brings attention to the broad involvement of liquid-liquid phase separation in all biological phenomena in a cell. The elucidation of the mechanism of autophagosome membrane expansion, which has been unknown for a long time in autophagy, is also expected to help to rapidly clarify the extremely complex mechanism of autophagy in the future. The elucidation of the mechanism will promote research for the treatment and prevention of various diseases related to autophagy. Further, the mechanism of membrane expansion by the cooperation of lipid transfer proteins and lipid scramblases discovered in this study is the first to be revealed not only in autophagy but also in cell biology, and is expected to contribute to the promotion of basic research in cell biology in general.