Screening of interacting proteins for IRE1 by yeast two-hybrid system
AIM: To screen the interacting proteins of IRE1 and to study its function and possible role in the apoptotic mechanism of hepatocytes. METHODS: Bait plasmids were constructed by cloning the 1-465 aa fragment in the N-termial part of hlRE1α and 468-977 aa fragment in the C-terminal part into pGBKT7 vector. Screening was conducted by sequential transformation of the bait plasmids and human-testis-cDNA library into AH109. Interaction was confirmed by screening in a series of different nutrition-deficient medium, X-α-GAL assays and PCR assays. Yeast two-hybrid retransformation experiment performed to verify the interactions. RESULTS: We successfully constructed the 2 bait plasmids and the expressions of the fusion-proteins were correct. Tests for autonomous activation and toxicity were negative. The numbers of the confirmed interactions were 8 for hIRE1α-N and 15 for MRE1α-C. Retransformation experiment verified an ER-resident apoptosis inducer SCOTIN, the transcript product of SHISA5 , as a binding partner of the C-terminal part of hIRE1α. CONCLUSION: The interaction between the C-terminal part of IRE1 and SCOTIN indicates a cross link between ER-stress-mediated and P53-mediated apoptofie signaling pathways.
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万方数据
维普
