In yeast, phosphatidylethanolamine is a target of the Atg8 modifier in 
ubiquitylation-like reactions essential for autophagy. Three human Atg8 (hAtg8) 
homologs, LC3, GABARAP, and GATE-16, have been characterized as modifiers in 
reactions mediated by hAtg7 (an E1-like enzyme) and hAtg3 (an E2-like enzyme) as 
in yeast Atg8 lipidation, but their final targets have not been identified. The 
results of a recent study in which COS7 cells were incubated with 
[14C]ethanolamine for 48 h suggested that phosphatidylethanolamine is a target 
of LC3. However, these results were not conclusive because of the long 
incubation time. To identify the phospholipid targets of Atg8 homologs, we 
reconstituted conjugation systems for mammalian Atg8 homologs in vitro using 
purified recombinant Atg proteins and liposomes. Each purified mutant Atg8 
homolog with an exposed C-terminal Gly formed an E1-substrate intermediate with 
hAtg7 via a thioester bond in an ATP-dependent manner and formed an E2-substrate 
intermediate with hAtg3 via a thioester bond dependent on ATP and hAtg7. A 
conjugated form of each Atg8 homolog was observed in the presence of hAtg7, 
hAtg3, ATP, and liposomes. In addition to phosphatidylethanolamine, in vitro 
conjugation experiments using synthetic phospholipid liposomes showed that 
phosphatidylserine is also a target of LC3, GABARAP, and GATE-16. In contrast, 
thin layer chromatography of phospholipids released on hAtg4B-digestion from 
endogenous LC3-phospholipid conjugate revealed that phosphatidylethanolamine, 
but not phosphatidylserine, is the predominant target phospholipid of LC3 in 
vivo. The discrepancy between in vitro and in vivo reactions suggested that 
there may be selective factor(s) involved in the endogenous LC3 conjugation 
system.