Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1.
The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha. As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.
Pubmed ID: 10187858 RIS Download
Adenosine Triphosphate | Amino Acid Sequence | Chromatography, Affinity | Cloning, Molecular | DNA-Binding Proteins | Fungal Proteins | HeLa Cells | Humans | I-kappa B Proteins | Ligases | Molecular Sequence Data | Molecular Weight | Protein Conformation | Recombinant Proteins | Repressor Proteins | SUMO-1 Protein | Saccharomyces cerevisiae Proteins | Sequence Homology, Amino Acid | Small Ubiquitin-Related Modifier Proteins | Ubiquitin-Activating Enzymes | Ubiquitin-Protein Ligases | Ubiquitins