Two conjugation systems and the Apg1 regulatory complex are required for vesicle formation. A: Apg12-Apg5 conjugation. Apg12 is activated by an ubiquitin activating enzyme E1 homologue, Apg7, through the formation of a thioester linkage through a C-terminal glycine of Apg12. The activated Apg12 is then transferred to the E2 enzyme Apg10. Finally, Apg10 transfers Apg12 to Apg5 resulting in the formation of a stable isopeptide bond between Apg12 and an internal lysine residue of Apg5. The Apg12-Apg5 conjugate binds to Apg16. Apg16 undergoes tetramerization resulting in a multi-conjugate complex that is required for autophagosome and Cvt vesicle formation. B: Aut7 lipid conjugation. Prior to Aut7 conjugation, the carboxyl-terminal arginine (R) residue of Aut7 is proteolytically removed by a cysteine protease, Aut2. The exposed glycine (G) residue of Aut7 is activated by the E1-like enzyme Apg7, and subsequently transferred to an E2 enzyme, Aut1. Aut7 is finally conjugated to PE by the formation of an amide bond between PE and Aut7. The resulting Aut7-PE conjugate allows Aut7 to be tightly associated with membrane that is involved in autophagosome and Cvt vesicle formation. A pool of Aut7 is subsequently cleaved from PE by the action of Aut2. C: The Apg1 regulatory complex. The Cvt pathway and autophagy are controlled by nutrient conditions through the Tor pathway. Under nutrient rich conditions, Tor kinase is active, and Apg13 is maintained in a hyperphosphorylated state resulting in a weak association with Apg1 kinase. Apg13 is only required for autophagy and physically associates with Vac8 that is only required for the Cvt pathway. Apg1 kinase also is phosphorylated and physically interacts with the Cvt pathway specific protein Cvt9 and the autophagy specific protein, Apg17. Apg17 associates with 2 additional Cvt specific proteins, Cvt13 and Cvt20, both of which contain PX domains that bind PtdIns(3)P. The phosphorylation state of Cvt13, Cvt20, and Apg17 is not known. Dephosphorylated Apg13 shows enhanced interaction with Apg1 under starvation conditions. Modified from Khalfan and Klionsky (56).