(1) First, SENPs process newly synthesized SUMO precursor into mature SUMO. (2) Then, SUMO's exposed di-glycine forms a thioester bond with the SAE2's catalytic cysteine in an ATP-dependent manner. (3) SUMO is then passed from the SAE1/SAE2 E1 activating heterodimer to the E2 conjugating enzyme UBC9, which also forms a thioester bond. (4) Substrates can be directly modified by E2-SUMO, but E3s might enhance conjugation rates by binding either E2-SUMO or substrates. (5) SENPs cleave the isopeptide bond and SUMO as well as the substrate are recycled. S: SUMO. ∼: thioester bond.