In vitro SUMO-1 modification requires two enzymatic steps, E1 and E2

The SUMO-1 has been identified as a protein that is highly similar to ubiqu itin and shown to conjugate to RanGAP1, PML, Sp200 and I kappa B alpha. The conjugation steps are thought to be similar to those of ubiquitination; an d human Ubc9, which is homologous to the E2 enzyme for the ubiquitin conjug ation step, was identified and shown to be necessary for the conjugation of SUMO-1 to its target protein. Other essential enzymes involved in this mod ification, however, remain to be clarified. Here we cloned human Sua1 (SUMO -1 activating enzyme) and hUba2, which are human homologs of yeast Saccharo myces cerevisiae Aos1 and Uba2, respectively. The recombinant proteins, Sua 1p and hUba2p, formed a complex. In this complex, hUba2 bound SUMO-1 and th is complex had the activity of the SUMO-1 activating enzyme, Furthermore, i n an in vitro system, RanGAP1 was modified by SUMO-1 in the presence of Sua 1p/Uba2p and hUbc9p, showing that the modification of SUMO-1 could be catal yzed by two enzyme steps, although ubiquitination usually requires three en zyme steps. (C) 1999 Academic Press.