UCSF

The endoplasmic reticulum (ER) is the site of folding and maturation for virtually all secreted and transmembrane proteins. When the ER folding capacity is overwhelmed, conserved signaling pathways in eukaryotes, collectively termed the unfolded protein response (UPR), sense this ER stress to reestablish proteostasis or initiate apoptosis in conditions of unresolved stress. The best studied of the three known UPR sensors, IRE1α, consists of an ER lumenal sensing domain, a single transmembrane helix followed by cytosolic kinase and ribonuclease (RNAse) effector domains. During ER stress, IRE1α activates by oligomerization, trans-autophosphorylation and subsequent allosteric RNase activation. Active IRE1α RNase initiates noncanonical splicing of its XBP1 mRNA substrate to allow the translation of active XBP1s transcription factors that in turn upregulate hundreds of genes to increase ER folding capacity and reduce folding load. In solution, IRE1α forms reversible oligomers that correlate with higher enzymatic activity. In ER-stressed cells, activated IRE1α dynamically clusters as discrete foci visible by fluorescence microscopy, which dissolves when stress is mitigated. This thesis presents three complementary approaches aimed to better understand the direct relationship between IRE1α oligomeric states and activity: 1. Characterizing the dynamic behavior of IRE1α molecules in mammalian cells during different UPR phases and corresponding IRE1α activation stages, 2. Solving the in situ structure of large IRE1α oligomers observed in stressed cells, and 3. Dissecting elements controlling IRE1α oligomerization. We discovered that: 1. Cellular IRE1α foci are entities with complex morphologies and dynamic behaviors consisting of two distinct populations of IRE1α: a diffusionally constrained core and a freely exchanging periphery. 2. In cells, IRE1α oligomers form ordered double helices contained in a newly described ER structure comprising extremely narrow (~28 nm) anastomosing membrane tubes. The arrangement of IRE1α molecules in this ER subdomain creates a positive feedback loop that sustains IRE1α activation and provides the structural basis for the two dynamically distinct populations observed. 3. A flexible linker region connecting IRE1α’s transmembrane helix to the effector kinase/RNase domains plays a major role in controlling IRE1α kinase/RNase domains activation and oligomerization in cells. Mutants carrying deletions or mutations within this region fail to form foci during stress and cannot mount an effective UPR. Taken together, this work bridges previous observations of IRE1α behaviors observed in vitro and IRE1α’s dynamic properties in cells to provide a more complete picture of why IRE1α oligomerization and activation are intricately linked.