Identification of the enzyme required for activation of the small ubiquitin-like modifier protein SUMO-1

Graham Duncan Kemp, JMP Desterro, MS Rodriguez, Ronald Thomas Hay

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295 Citations (Scopus)

Abstract

The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), I kappa B alpha, 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 I kappa B alpha. As SAE1/SAE2, Ubch9, SUMO-1, and I kappa B alpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of I kappa B alpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.

Original languageEnglish
Pages (from-to)10618-10624
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Publication statusPublished - 9 Apr 1999

Keywords

  • KAPPA-B-ALPHA
  • NUCLEAR-PORE COMPLEX
  • AFFINITY PURIFICATION
  • CONJUGATING ENZYME
  • SYSTEM
  • DEGRADATION
  • PML
  • TERMINUS
  • HOMOLOG
  • RANGAP1

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