Molecular mechanism of autophagy. Autophagy initiation starts with activation of the ULK1/2 kinase complex, which also includes ATG13 and FIP200. mTOR suppresses the activity of this complex by phosphorylating ULK1 and ATG13 on “negative” sites. This complex then becomes active after AMPK activation and mTOR inhibition, for example, during starvation or treatment with rapamycin. Phosphorylated and now-active ULK1 promotes phosphorylation of ATG13 and FIP200 and dissociates from mTOR. The PI3K-III VPS34 is critical for further autophagosome formation. VPS34 forms a complex with VPS15, UVRAG, AMBRA1, and BECLIN1. This complex can be inhibited by the antiapoptotic protein BCL-2, which can interact with BECLIN1 through the BH3 domain in BECLIN1. Autophagosome completion is mediated by the ATG8 (LC3)-PE and ATG12/ATG5 conjugation systems. These systems perform the lipid modification of LC3-I, leading to LC3-II-PE binding to the autophagosomal membrane (see “Autophagosome formation”). The conversion of LC3-I to LC3-II is commonly used to monitor autophagy by various assays. Completed autophagosomes contain materials such as proteins and organelles that can be digested after autophagosome fusion with lysosomes. Lysosomes have low pH and an abundance of pH-sensitive enzymes that can break down waste materials and cellular debris. The autolysosome degradation can therefore remove unwanted materials such as damaged proteins in addition to providing the cell with new building blocks for cellular maintenance. The mTOR inhibitor rapamycin can be used to promote autophagy induction. VPS34 inhibitors, such as 3-MA and wortmannin, can be used to inhibit the early stages of autophagy. Bafilomycin A₁, CQ, HCQ, and NH₄Cl, which inhibit the fusion of autophagosomes and lysosomes, can be used to inhibit autophagy at later stages.