The ADP-binding kinase region of Ire1 directly contributes to its responsiveness to endoplasmic reticulum stress

Quynh Giang Le,Kenji Kohno,Yukio Kimata,Shigeto Fukunaka,Yuki Ishiwata-Kimata,Thi Huong Phuong

doi:10.1038/s41598-021-83890-x

Abstract

Upon endoplasmic-reticulum (ER) stress, the ER-located transmembrane protein, Ire1, is autophosphorylated and acts as an endoribonuclease to trigger the unfolded protein response (UPR). Previous biochemical studies have shown that Ire1 exhibits strong endoribonuclease activity when its cytosolic kinase region captures ADP. Here, we asked how this event contributes to the regulation of Ire1 activity. At the beginning of this study, we obtained a luminal-domain mutant of Saccharomyces cerevisiae Ire1, deltaIdeltaIIIdeltaV/Y225H Ire1, which is deduced to be controlled by none of the luminal-side regulatory events. ER-stress responsiveness of deltaIdeltaIIIdeltaV/Y225H Ire1 was largely compromised by a further mutation on the kinase region, D797N/K799N, which allows Ire1 to be activated without capturing ADP. Therefore, in addition to the ER-luminal domain of Ire1, which monitors ER conditions, the kinase region is directly involved in the ER-stress responsiveness of Ire1. We propose that potent ER stress harms cells’ “vividness”, increasing the cytosolic ADP/ATP ratio, and eventually strongly activates Ire1. This mechanism seems to contribute to the suppression of inappropriately potent UPR under weak ER-stress conditions.

Highlights

Upon endoplasmic-reticulum (ER) stress, the ER-located transmembrane protein, Ire[1], is autophosphorylated and acts as an endoribonuclease to trigger the unfolded protein response (UPR)
Since the intracellular activity of Ire[1] is monitored using a UPR element (UPRE)-based reporter, from which β-galactosidase is expressed under the control of the U PRE25,26, we reproduced these findings through the UPRE-lacZ reporter assay
Based on our observations presented far, we speculate that ΔIΔV/Y225H Ire[1] and ΔIΔIIIΔV/Y225H Ire[1] are regulated by none of the previously known regulatory events on the luminal side

Summary

Introduction

Upon endoplasmic-reticulum (ER) stress, the ER-located transmembrane protein, Ire[1], is autophosphorylated and acts as an endoribonuclease to trigger the unfolded protein response (UPR). ER-stress responsiveness of deltaIdeltaIIIdeltaV/Y225H Ire[1] was largely compromised by a further mutation on the kinase region, D797N/K799N, which allows Ire[1] to be activated without capturing ADP. In addition to the ER-luminal domain of Ire[1], which monitors ER conditions, the kinase region is directly involved in the ER-stress responsiveness of Ire[1]. We propose that potent ER stress harms cells’ “vividness”, increasing the cytosolic ADP/ATP ratio, and eventually strongly activates Ire[1] This mechanism seems to contribute to the suppression of inappropriately potent UPR under weak ER-stress conditions. According to various experimental approaches taken by us and o thers[12,13,14], the luminal domain of S. cerevisiae Ire[1] has been shown to carry two intrinsically disordered segments, namely Subregions I and V, and Scientific Reports | (2021) 11:4506

Methods

Results

Conclusion