Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1

We used cysteine crosslinking to characterize the configuration of the transmembrane region of the unfolded protein response (UPR) transducer Ire1 from baker's yeast during different forms of stress. We find that different forms of ER stress converge in a single configuration of Ire1. The functionality of the individual single-cysteine variants of Ire1 was characterized by microscopy, cell-based ER stress resistance assays, and qPCR to characterize the degree of HAC1 mRNA splicing and the induction of UPR target gens..本研究采用半胱氨酸交联技术，解析了酿酒酵母来源的未折叠蛋白反应（Unfolded Protein Response, UPR）转导蛋白Ire1的跨膜区域在不同内质网应激条件下的构象。研究发现，不同类型的内质网应激最终会使Ire1收敛至同一种构象。本研究通过显微观测、基于细胞的内质网应激抗性实验以及定量聚合酶链反应（qPCR），对Ire1的各单半胱氨酸变体的功能进行了表征；其中qPCR用于检测HAC1 mRNA的剪接水平以及UPR靶基因的诱导表达情况。