Abstract
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.
Highlights
From the ‡Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan, §AGENE Research Institute, 200 Kajiwara, Kamakura 247, Japan, and ¶The Picower Institute for Medical Research, Manhasset, New York 11030
The interaction of Werner’s syndrome (WS) encodes a protein (Wrn) and SUMO-1 was abolished by deleting the Gly residue, indicating that the interaction of Wrn and SUMO-1 is mediated by yeast Ubc9 (yUbc9) in the two-hybrid system
We screened 2.3 ϫ 106 transformants derived from a mouse lymphoma cDNA library and identified 50 positive clones, which were categorized into 3 different groups, Whip (20 clones), which encodes a novel protein, Ubc9 (29 clones), and SUMO-1 (1 clone)
Summary
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. To obtain further insight into the process in which Wrn is involved, we tried to isolate cDNAs encoding proteins that interacted with mouse Wrn by using the yeast two-hybrid system. Construction of pGBT9-yUBC9 and pGAD424-yUBC9 —Budding yeast genomic UBC9 DNA was amplified by PCR and was inserted into pGEM-T-Easy vector.
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