The FAP motif within human ATG7, an autophagy-related E1-like enzyme, is essential for the E2-substrate reaction of LC3 lipidation.

ATG7 is an autophagy-related E1-like enzyme that is essential for two ubiquitination-like reactions, ATG12-conjugation and LC3-lipidation. The existence of functional sequences at the amino-terminal region of human ATG7 remains uncertain. Mutational analyses of ATG7 revealed that both mutant ATG7ΔFAP lacking the FAP motif and ATG7FAPtoDDD, in which the Phe15-Ala16-Pro17 sequence was changed to Asp-Asp-Asp, could not complement defects in endogenous ATG12-conjugation and LC3-lipidation when expressed in Atg7-deficient mouse embryonic fibroblasts (MEFs). However, wild-type ATG7 complemented the defects in these cells. Overexpression of GFP-ATG10 and GFP-ATG12 rescued a defect in ATG12-conjugation in Atg7-deficient MEFs expressing mutant ATG7ΔFAP and ATG7FAPtoDDD, whereas overexpression of all ATG proteins related to ATG12-conjugation and LC3-lipidation could not rescue a defect in LC3-lipidation in Atg7-deficient MEFs expressing these ATG7 mutants. Both ATG7ΔFAP and ATG7FAPtoDDD mutants showed severe defects in the formation of an E2-substrate intermediate of ATG3 with LC3 in LC3-lipidation, but were able to form an E1-substrate intermediate of ATG7 with LC3 and the E1- and E2-substrate intermediates in ATG12-conjugation with reduced efficiency. These ATG7 mutants could also form the ATG12-ATG3 conjugate. Co-immunoprecipitation experiments revealed that the FAP motif of ATG7 is essential for the interaction of ATG7 with ATG3, but not for ATG7-homodimerization. These results indicated that the FAP motif of ATG7 is indispensable for formation of the ATG3-LC3 E2-substrate intermediate through the interaction of ATG7 with ATG3.