Autophagy Related Protein 3 (ATG3)

ATG3 formed an enzyme-substrate intermediate via a thiol ester bond that showed rapid turnover. Replacement of the active-site cysteine of ATG3 with serine (C264S) resulted in the formation of a stable O-ester bond that allowed visualization of stable intermediates. Overexpression of ATG7 enhanced the formation of stable E2-substrate complexes between ATG3-C264S and each of the Apg8 homologs. MAPLC3 was the preferred substrate, and the reaction with MAPLC3 occurred predominantly in the cytosol. Coimmunoprecipitation of ATG7 with ATG3 indicated that ATG3 forms an E1-E2 complex with ATG7, similar to the yeast Apg3-Apg7 complex. Overexpression of ATG3 facilitated formation of the ATG12-ATG5 conjugate, suggesting that ATG3 cross-talks with the ATG12 conjugation system.