Purification and activity assays of the catalytic domains of the kinase/endoribonuclease Ire1p from Saccharomyces cerevisiae.

Ire1p is a single-spanning transmembrane protein of the endoplasmic reticulum (ER) of all eukaryotic cells. A bifunctional enzyme exhibits both kinase and site-specific endoribonuclease activities. Work with the yeast Saccharomyces cerevisiae showed that Ire1p provides a key regulatory switch during an intracellular signaling pathway that originates in the lumen of the ER. When unfolded proteins accumulate in the ER, a signal is sent to induce a transcriptional program, termed the unfolded protein response or UPR that causes an increase in the protein-folding capacity of the ER. An accumulation of unfolded proteins is initially sensed by the ER–lumenal domain of Ire1p by an unknown mechanism. Ire1p molecules are then thought to laterally oligomerize in the plane of the membrane, which leads to trans-autophosphorylation of their kinase domains and concomitant activation of an endoribonuclease activity. This chapter describes the overexpression in two different systems, purification, and activity assays for fusion proteins containing the cytosolic domains of Ire1p from S. cerevisiae. All expressed proteins truncate Ire1p just after the transmembrane region and hence contain the linker, kinase, and presumed nuclease domains.