Covalent modification of the Werner's syndrome gene product with the ubiquitin-related protein, SUMO-1.
Werner's syndrome is a potential model of accelerated human aging. The gene responsible for Werner's syndrome encodes a protein that has a helicase domain homologous to Escherichia coli RecQ. To identify binding partners that regulate the function in concert with Wrn, we screened for proteins using the yeast two-hybrid system with mouse Wrn as bait and found three. One was a novel protein, and the other two were mouse Ubc9 and SUMO-1. Ubc9 also interacted with the mouse homologue of the Bloom's syndrome gene product, another eukaryotic RecQ-type helicase, but not mouse DNA helicase Q1/RecQL (RecQL1). Deletion experiments indicated that both proteins interacted with the N-terminal segment of Wrn (amino acid 272-514). The interaction between Wrn and SUMO-1 was weaker than that between Wrn and Ubc9. Positive interaction was observed in the heterogeneous combination of Wrn and yeast Ubc9 (yUbc9), as well as yUbc9 and SUMO-1, in the two-hybrid system. The interaction between yUbc9 and SUMO-1 was abolished by deleting the C-terminal Gly residue of SUMO-1, which is reportedly required for the formation of Ubc9-SUMO-1 thioester linkage. The interaction of Wrn and SUMO-1 was also abolished by deleting the Gly residue, indicating that the interaction of Wrn and SUMO-1 is mediated by yUbc9 in the two-hybrid system. Finally, we confirmed by immunoblotting with an anti-SUMO-1 antibody that Wrn was covalently attached with SUMO-1.
Kawabe
Y
Y
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Seki
M
M
Seki
T
T
Wang
W S
WS
Imamura
O
O
Furuichi
Y
Y
Saitoh
H
H
Enomoto
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001816:Bloom Syndrome / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D006801:Humans; D008025:Ligases / Q000378:metabolism; D051379:Mice; D053484:RecQ Helicases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025842:SUMO-1 Protein; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*; D014898:Werner Syndrome / Q000235:genetics; D000071657:Werner Syndrome Helicase; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; D005090:Exodeoxyribonucleases; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; C506725:RECQL protein, human; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9