: The macroautophagy/autophagy machinery has two ubiquitin-like (UBL) conjugation systems. The Atg8/MAP1LC3/GABARAP (yeast/human) and Atg12/ATG12 proteins are UBL substrates for Atg7/ATG7, a non-canonical E1 enzyme, that thioesterifies its substrates; however, autophagy requires a much greater amount of conjugated Atg8 (Atg8–PE) than Atg12 (Atg12–Atg5). Exactly how Atg7/ATG7 distinguishes between its two substrates to facilitate this differential biogenesis remains elusive. Here, analyses of recombinant complexes of yeast proteins reveal that the N termini of Atg8 and Atg12 are structural determinants for conjugation to Atg7, but play no role in conjugation to Atg3 or Atg10, non-canonical E2 enzymes. The disordered N terminus of Atg12 is a protector of the Atg12 C terminus and a negative regulator of Atg7–Atg12 conjugation and autophagy, whereas the N-terminal helical domain in Atg8 promotes autophagy and has a high avidity to Atg7. We show that balanced autophagy requires different specific N termini attached to the UBL domains.