Intragenic complementation in the protein kinase domain in Ire1

In eukaryotic cells, secretory and transmembrane proteins fold in the endoplasmic reticulum (ER) before they exit the ER. Accumulation of unfolded proteins activatesIre1 to promote the unfolded protein response (UPR). In the yeast Saccharomyces cerevisiae, activation of Ire1 leads tosplicing of the mRNA for the transcription factor Hac1. Wehave usedb-galactosidase reporter assaysto demonstrateexpression of a UPRE-lacZreporter genethat is positively regulated by the Ire1–Hac1 signalling pathway. In this study, the protein kinase domain was subjected to mutations to alter the catalytic aspartate D797 and lysine K799, which interacts with the terminal phosphate group of ATP, to alanine. Also, point mutations in the Mg+2coordinating loop converted asparagine N802 and aspartic acid D828 to alanine. The expression of theUPRE-lacZreporter gene in all single mutants K799, N802 and D797 was decreased compared to WT-Ire1. Under conditions of various ER stress inducing drugs the ability of the D797A mutant strain to survive was reduced in growth assays compared to other mutants in theMg+2 coordinating loop and catalytic domain. D797-Ire1 mutant has less inb-galactosidase activity, survival of ER stress and HAC1 splicing. Expression of Ire1 carrying double mutations D797A-N802A increasedb-galactosidase activity significantly and restored growth compared to the single mutant D797A-Ire1. WT and protein kinase mutants express to the same level. This study suggeststhat introducing a second mutation such as K799A-Ire1 or N802A-Ire1 to the single mutant D797A-Ire1, restores signaling activity of Ire1.