Fujioka Yuko, Tsuji Takuma, Kotani Tetsuya, Kumeta Hiroyuki, Kakuta Chika, Shimasaki Junko, Fujimoto Toyoshi, Nakatogawa Hitoshi, Noda Nobuo N
Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan.
Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Japan.
Nat Struct Mol Biol. 2025 Sep 16. doi: 10.1038/s41594-025-01678-3.
Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the preautophagosomal structure (PAS) in Saccharomyces cerevisiae, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12-Atg5-Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets through specific Atg12-Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution demonstrates that E3-enriched early PAS droplets promote Atg8 lipidation and that Atg8 coating of the vesicle membrane is both necessary and sufficient for their condensation into the droplets. These data suggest that the PAS functions as an efficient production site for lipidated Atg8 and pools membrane seeds to drive autophagosome formation.
在饥饿状态下，自噬起始因子Atg1复合物会发生相分离，从而在酿酒酵母中组织前自噬体结构（PAS），自噬体的形成被认为是从此处开始的。然而，PAS液滴的生理作用仍不清楚。在这里，我们表明核心Atg蛋白被招募到早期PAS液滴中，这些液滴是由Atg1复合物在体外以不同效率相分离形成的。用于Atg8脂化的Atg12-Atg5-Atg16 E3连接酶复合物通过特定的Atg12-Atg17相互作用最有效地浓缩在液滴中，这对该E3复合物在体内靶向PAS也很重要。体外重建表明，富含E3的早期PAS液滴促进Atg8脂化，并且囊泡膜上的Atg8包被对于它们浓缩成液滴既必要又充分。这些数据表明，PAS作为脂化Atg8的高效生产位点，并汇集膜种子以驱动自噬体形成。
