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.
Desterro
J M
JM
School of Biomedical Science, University of St. Andrews, St. Andrews, Fife KY169ST Scotland.
Rodriguez
M S
MS
Kemp
G D
GD
Hay
R T
RT
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000072000:NF-KappaB Inhibitor alpha; D011487:Protein Conformation; D011994:Recombinant Proteins; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D025842:SUMO-1 Protein; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C111706:RUB1 protein, S cerevisiae; D011994:Recombinant Proteins; D012097:Repressor Proteins; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C470301:UBA2 protein, human; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; C470299:SAE1 protein, human; D044764:Ubiquitin-Activating Enzymes