Table 1.

A brief summary of the studies on in vitro reconstitution of autophagy

Reconstitution		Main conclusion	Citation
Protein–membrane interaction	Atg8/LC3 lipidation	(1) Reconstitution of Atg8/LC3 lipidation with purified proteins and liposomes demonstrated that Atg7, Atg3, and Atg12−Atg5 act as E1, E2, and E3-like enzymes for conjugating Atg8/LC3 to PE.	(46–49)
		(2) Reconstitution of Atg8 lipidation with different kinds of liposomes indicated that Atg16 selectively promotes Atg8 lipidation on less curved membrane by facilitating the membrane targeting of Atg12−Atg5.	(50)
		(3) Reconstitution of LC3 lipidation with different sized liposomes indicated that ATG3 prefers curved membrane for LC3 lipidation.	(51)
	Membrane targeting of autophagic factors	(1) Sedimentation assays determined that Atg14 is recruited to membrane that is highly curved and enriched in PI3P and PI(4,5)P2, when mTORC1 is inhibited.	(52,53)
		(2) Cell-free reconstitution and membrane floatation indicate a starvation-induced recruitment of ATG14 to the ERGIC membrane.	(54)
Autophagic membrane remodeling	Autophagosome membrane origin	(1) Reconstitution of autophagic signal-regulated LC3 lipidation in a cell-free system and membrane fractionation identify the ERGIC as an essential membrane source of the autophagosome.	(54)
	Autophagosome precursor generation	(1) Reconstitution of autophagic signal-regulated early autophagic precursor generation identifies a PI3K-induced switch of COPII location from the ER to the ERGIC as an essential mechanism for mobilizing the donor membrane for early autophagic precursor biogenesis.	(55)
	Membrane tethering and fusion	(1) Atg8 family proteins, either lipidated in vitro or chemically conjugated to PE, are capable of tethering vesicles and promote membrane fusion.	(56–59)
		(2) Structural and binding analyses indicated that Atg1/Atg13/Atg17/Atg29/Atg31 complex and TRAPPIII complex may be capable of tethering autophagic vesicles.	(60,61)
		(3) Cell-free reconstitution indicated that the autophagosome can fuse with endosomes and lysosomes and that this is dependent on cytosolic factors.	(62)
		(4) Liposome tethering and fusion assay indicates that ATG14 is capable of tethering membrane and mediating membrane hemifusion and fusion that is important for autophagosome and endolysosome fusion.	(63)
	Phagophore shape and asymmetry	(1) Reconstitution of the Atg12−Atg5/Atg16 assembly on GUVs with lipidated Atg8 indicated that Atg16 anchors Atg12−Atg5 and lipidated Atg8 by forming a tetramer, which facilitates the formation of a two-dimensional meshwork likely to be a scaffold shaping the phagophore.	(64)
		(2) Cargo adaptor Atg32 competes with the Atg12−Atg5/Atg16 complex to associate with Atg8, which could be an explanation for the asymmetrical localization of cargo and autophagic factors on the concave and convex face of the phagophore.	(64)
	Lysosome reformation	(1) Cell-free reconstitution pinpointed the role of clathrin and clathrin adaptors and PI(4,5)P2 in lysosome reformation after autophagy.	(10)
Selective Autophagy	Localized signal activation	(1) Cell-free reconstitution with a genetically engineered Atg1 indicates that a regulatory chain from cargo, adaptor and scaffold activates Atg1 through direct protein interaction, which accounts for the local activation of autophagy around the cargo during selective autophagy.	(65)
	Efficient cargo recognition	(2) The yeast cargo adaptor Atg19 has multiple AIMs. Reconstitution of membrane and cargo binding indicates that cargo binding enables the availability of AIMs to interact with lipidated Atg8, which enhances the recruitment of autophagic membranes.	(66)
		(3) The mammalian cargo adaptor P62 has one LIR. Reconstitution of membrane and cargo binding indicates that oligomerization enables multiple LIRs in one protein complex. This strengthens the binding of P62 to LC3 and ubiquitinated cargos as a mechanism to enhance the recruitment of autophagic membranes to the cargo.	(67)