2ER MEMBRANE AND LUMENAL PROTEIN INTERACTION YEAST TWO-HYBRID ASSAY

摘要

The present invention relates to a new yeast two-hybrid systemfor the detection of interactions between membrane proteins and lumenalproteins of the endoplasmic reticulum. The yeast two-hybrid system of thepresent invention uses the IRE1 gene of yeast which is a key element inthe endoplasmic reticulum unfolded protein response to signal theinteraction between proteins within the endoplasmic reticulum. The IRE1gene codes for a type 1 membrane protein Ire1p, that has a kinase in thecytosolic domain and it signals the presence of unfolded proteins in the ERby oligomerization and transphosphorylation, this in turn activates aRNaseL that has as its unique (so far) substrate the HAC1 messengerRNA, that codes for the transcription factor that binds to the unfoldedprotein response element and upregulates transcription of ER proteinrequired for folding proteins within the ER. The yeast two-hybrid systemof the present invention uses in-reading frame fusions of ER proteins tothe N-terminal "protein sensing domain" of Ire1p to detect their interactionusing Ire1p dimerization and the unfolded protein response system as aread out. This readout is made simpler by the use of reporter genesystems making the system suitable for mass screening of ER proteininteractions.