Direct Binding to Unfolded Proteins Activates Ire1 and the Unfolded Protein Response

Secreted and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded, nascent polypeptides. Before they continue along the secretory pathway to their final destination, these proteins must fold into their proper structure. The ER is the primary site of quality control for these proteins; terminally misfolded proteins are degraded, while properly folded proteins are packaged into vesicles. This quality control ensures proper folding, and thus proper function, for all of the proteins that mediate cell-environment interactions. However, the mechanisms required for quality control can also cause an accumulation of unfolded proteins when environmental stresses, developmental programs, or drug treatments disrupt protein folding. Cells facing such ER stress activate a signaling pathway, the Unfolded Protein Response (UPR), designed to restore protein folding homeostasis.In S. cerevisiae, the UPR is activated by the transmembrane protein, Ire1, which initiates a transcriptional program to increase the size and capacity of the ER. Ire1 consists of a stress-sensing lumenal domain that oligomerizes in response to ER stress and cytoplasmic domains that transmit signals to the nucleus and cytoplasm. Ire1 and its activation by oligomerization are conserved throughout the Metazoa. Despite the ubiquity of the UPR pathway and its relevance to human disease, the mechanism of Ire1 stress-sensing and subsequent oligomerization were largely unknown. In Chapter Two, we provide biochemical evidence that unfolded proteins are ligands for Ire1 activation by showing that Ire1 forms a complex in vivo with a model misfolded protein, binds short peptide proxies in vitro, and that peptide binding causes Ire1 oligomerization. Chapter Three extends our findings with Ire1 cLD from S. cerevisiae to the Ire1 lumenal domain from Mus musculus. Although the crystal structure of MmIre1 LD revealed a narrower binding groove, we found that MmIre1 LD can also bind peptides in vitro, but with a different binding preference than ScIre1 cLD. In Chapter Four, we crystallized the lumenal domain of the Ire1 homolog from a thermophilic yeast, Chaetomium thermophilum. This structure reveals several intriguing new interactions within the crystal lattice, which may be important for Ire1 activation