Abstract
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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adenosine Triphosphate / metabolism
-
Amino Acid Sequence
-
Chromatography, Affinity
-
Cloning, Molecular
-
DNA-Binding Proteins / metabolism
-
Fungal Proteins / chemistry
-
Fungal Proteins / metabolism
-
HeLa Cells
-
Humans
-
I-kappa B Proteins*
-
Ligases / genetics
-
Ligases / isolation & purification*
-
Ligases / metabolism
-
Molecular Sequence Data
-
Molecular Weight
-
NF-KappaB Inhibitor alpha
-
Protein Conformation
-
Recombinant Proteins
-
Repressor Proteins / chemistry
-
Repressor Proteins / metabolism
-
SUMO-1 Protein
-
Saccharomyces cerevisiae Proteins*
-
Sequence Homology, Amino Acid*
-
Small Ubiquitin-Related Modifier Proteins*
-
Ubiquitin-Activating Enzymes
-
Ubiquitin-Protein Ligases
-
Ubiquitins / metabolism*
Substances
-
DNA-Binding Proteins
-
Fungal Proteins
-
I-kappa B Proteins
-
NFKBIA protein, human
-
RUB1 protein, S cerevisiae
-
Recombinant Proteins
-
Repressor Proteins
-
SUMO-1 Protein
-
SUMO2 protein, human
-
Saccharomyces cerevisiae Proteins
-
Small Ubiquitin-Related Modifier Proteins
-
UBA2 protein, human
-
Ubiquitins
-
NF-KappaB Inhibitor alpha
-
Adenosine Triphosphate
-
Ubiquitin-Protein Ligases
-
Ligases
-
SAE1 protein, human
-
Ubiquitin-Activating Enzymes
Associated data
-
GENBANK/AF110956
-
GENBANK/AF110957