(308h) Studies of IRE1alpha Transmembrane Domain Dimerization in Lipid Bilayers

IRE1α (inositol-requiring enzyme 1α) is an integral membrane protein with an N-terminal luminal domain and a C-terminal cytosolic domain, which are connected by a single transmembrane (TM) helix.  As one of the three transmembrane sensor proteins, IRE1α propagates the signal from the ER (endoplasmic reticulum) lumen to the cytosol. It has been known that the ER luminal domain of IRE1α is maintained in its inactive monomeric state by interacting with ER chaperone, Bip. However, upon ER stress, Bip is able to release from the luminal domain, allowing the luminal domain to dimerise or oligomerize. This promotes the dimerization of the TM domains and cytosolic domains, which results in activation of the cytosolic domain and induction of several signaling pathways to relieve ER stress. However, the molecular mechanism of signal transduction across the membrane remains unknown. In addition, although the TM domains are expected to play an important role in the dimerization process, the contribution of IRE1α TM domain to dimer stability has not been investigated. We address whether the TM domains are able to form dimers/oligomers in a membrane-like environment. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining analyses show that three states (monomers, dimers, and high-oligomers) of TM domain peptides exist in SDS micelles, even in the absence of both the luminal and cytosolic domains. Circular Dichroism (CD) spectra suggested that IRE1α TM domain peptide is helical in SDS micelles and in liposome. We performed FRET (Fӧrster resonance energy transfer) using FITC-labeled IRE1α TM peptide (donor) and Rhodamine-labeled IRE1α TM peptide (acceptor). FRET results suggest that IRE1α TM domain forms dimers in lipid bilayers. Since it is well known that palmitic acid (C16-saturated fatty acid) induces ER stress by activating IRE1α protein, using FRET energy analyses, we further determine whether palmitic acid is able to promote dimerization of IRE1α TM peptide in liposomes. Therefore, the results provide the first evidence that the IRE1α TM domains are capable of significant protein–protein interactions to thereby contribute to ER stress-signaling propagation.