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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

Authors

  • Desterro JM
  • Rodriguez MS
  • Kemp GD
  • Hay RT

Journal

The Journal of biological chemistry

Publication Data

April 9, 1999

Associated Grants

None

Mesh Terms

  • 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