Cysteine crosslinking in native membranes establishes the transmembrane architecture of Ire1

Abstract

36The endoplasmic reticulum (ER) is a key organelle of membrane biogenesis and 37 crucial for the folding of both membrane and secretory proteins. Stress sensors of the 38 unfolded protein response (UPR) monitor the unfolded protein load in the ER and 39 convey effector functions for the maintenance of ER homeostasis. More recently, it 40 became clear that aberrant compositions of the ER membrane, referred to as lipid 41 bilayer stress, are equally potent activators of the UPR with important implications in 42 obesity and diabetes. How the distinct signals from lipid bilayer stress and proteotoxic 43 stress are processed by the highly conserved UPR transducer Ire1 remains unknown. 44Here, we have generated a functional, cysteine-less variant of Ire1 and performed 45 systematic cysteine crosslinking experiments to establish the transmembrane 46 architecture of signaling-active clusters in native membranes. We show that the 47 transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped 48 configuration and that this configuration is independent of the primary cause for ER 49 stress. Based on these findings, we propose that different forms of stress converge in 50 a single, signaling-active conformation of Ire1.