C - Chemistry – Metallurgy – 12 – Q
Patent
C - Chemistry, Metallurgy
12
Q
C12Q 1/68 (2006.01) C12N 1/16 (2006.01) C12N 15/10 (2006.01) G01N 33/566 (2006.01)
Patent
CA 2358758
The present invention relates to a new yeast two-hybrid system for the detection of interactions between membrane proteins and lumenal proteins of the endoplasmic reticulum. The yeast two-hybrid system of the present invention uses the IRE1 gene of yeast which is a key element in the endoplasmic reticulum unfolded protein response to signal the interaction between proteins within the endoplasmic reticulum. The IRE1 gene codes for a type 1 membrane protein Ire1p, that has a kinase in the cytosolic domain and it signals the presence of unfolded proteins in the ER by oligomerization and transphosphorylation, this in turn activates a RNaseL that has as its unique (so far) substrate the HAC1 messenger RNA, that codes for the transcription factor that binds to the unfolded protein response element and upregulates transcription of ER protein required for folding proteins within the ER. The yeast two-hybrid system of the present invention uses in-reading frame fusions of ER proteins to the N-terminal "protein sensing domain" of Ire1p to detect their interaction using Ire1p dimerization and the unfolded protein response system as a read out. This readout is made simpler by the use of reporter gene systems making the system suitable for mass screening of ER protein interactions.
Bergeron John J. M.
Jansen Gregor
Pelletier Marc F.
Thomas David Y.
National Research Council Of Canada
Ogilvy Renault Llp/s.e.n.c.r.l.,s.r.l.
University Mcgill
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