IntroductionAutophagy is triggered by various stresses such as nutrient starvation and infection, leading to rapid formation of an isolation membrane (IM) that engulfs cytoplasmic components. This IM closes into a double-membraned autophagosome. The Atg2–Atg18 complex localizes to the IM through phosphatidylinositol 3-phosphate (PI3P) binding and also associates with the ER and is thought to transport lipids from the ER to the IM, driving IM expansion1). However, the exact contribution of Atg2-mediated lipid transfer to autophagosome assembly and its molecular mechanisms remain uncertain. In order to elucidate the lipid transfer mechanism, we performed in vitro reconstitution assays using synthetic liposomes.We also embarked on another project. Autophagy is suppressed during sleep in Drosophila2), but how it relates to sleep is unclear. Nemuri (Nur), expressed in Drosophila upon bacterial infection, promotes both sleep and bacterial killing3). Since autophagy also destroys bacteria, the interplay among these processes is complex. To probe these relationships, we focused on Nur’s antibacterial activity and studied the molecular mechanisms.Experimental MethodsExpression and purification of Atg2 and Atg18The purification of Atg2 and Atg18 was performed according to previously published protocols1). Briefly, Atg2 and Atg18 proteins were expressed in BJ3505 yeast cells lacking endogenous atg2 and atg18 genes transformed with pRS426-based plasmids. After disruption of collected yeast cells using zirconia beads, the lysate was subjected to centrifugation. For purification of Atg2, the supernatant was subjected to HisPur Ni-NTA resin (Thermo Fisher Scientific). After elution of the protein using 50 mM HEPES (pH 8.0), 500 mM NaCl, 300 mM imidazole, and 10% glycerol, the eluate was dialyzed against buffer G consisting of 100 mM Tris-HCl (pH 8.0), 500 mM NaCl and 10% glycerol and then subjected to a Strep-Tactin Superflow high capacity resin column (IBA Lifesciences). After elution from the resin using buffer G supplemented with 10 mM desthiobiotin, the eluate was dialyzed against 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1mM DTT, concentrated, and stored at -80°C until use. For purification of Atg18, the supernatant of the lysate was supplemented with avidin at approximately 30 μg ml-1 final concentration at 4°C and then was subjected to Strep-Tactin XT high capacity Moynul Hasan2022. 8 ～ 2024. 7Nobuo Noda, ProfessorInstitute for Genetic Medicine, Hokkaido University― 251 ―In vitro reconstitution of phagophore expansion and sealing using purified proteins and liposomes
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