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

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          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.

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          Most cited references 36

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          Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase.

          The transcription of genes encoding soluble proteins that reside in the endoplasmic reticulum (ER) is induced when unfolded proteins accumulate in the ER. Thus, an intracellular signal transduction pathway must exist that mediates communication between the ER lumen and the nucleus. We have identified a gene in S. cerevisiae, IRE1, that is required for this pathway: ire1- mutants cannot activate transcription of KAR2 and PDI1, which encode the ER resident proteins BiP and protein disulfide isomerase. Moreover, IRE1 is essential for cell viability under stress conditions that cause unfolded proteins to accumulate in the ER. IRE1 encodes a transmembrane serine/threonine kinase that we propose transmits the unfolded protein signal across the ER or inner nuclear membrane. IRE1 is also required for inositol prototrophy, suggesting that the induction of ER resident proteins is coupled to the biogenesis of new ER membrane.
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            Signal integration in the endoplasmic reticulum unfolded protein response.

            The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways - cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.
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              On the mechanism of sensing unfolded protein in the endoplasmic reticulum.

              Unfolded proteins in the endoplasmic reticulum (ER) activate the ER transmembrane sensor Ire1 to trigger the unfolded protein response (UPR), a homeostatic signaling pathway that adjusts ER protein folding capacity according to need. Ire1 is a bifunctional enzyme, containing cytoplasmic kinase and RNase domains whose roles in signal transduction downstream of Ire1 are understood in some detail. By contrast, the question of how its ER-luminal domain (LD) senses unfolded proteins has remained an enigma. The 3.0-A crystal structure and consequent structure-guided functional analyses of the conserved core region of the LD (cLD) leads us to a proposal for the mechanism of response. cLD exhibits a unique protein fold and is sufficient to control Ire1 activation by unfolded proteins. Dimerization of cLD monomers across a large interface creates a shared central groove formed by alpha-helices that are situated on a beta-sheet floor. This groove is reminiscent of the peptide binding domains of major histocompatibility complexes (MHCs) in its gross architecture. Conserved amino acid side chains in Ire1 that face into the groove are shown to be important for UPR activation in that their mutation reduces the response. Mutational analyses suggest that further interaction between cLD dimers is required to form higher-order oligomers necessary for UPR activation. We propose that cLD directly binds unfolded proteins, which changes the quaternary association of the monomers in the membrane plane. The changes in the ER lumen in turn position Ire1 kinase domains in the cytoplasm optimally for autophosphorylation to initiate the UPR.
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                Author and article information

                Contributors
                kimata@bs.naist.jp
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                24 February 2021
                24 February 2021
                2021
                : 11
                Affiliations
                [1 ]GRID grid.260493.a, ISNI 0000 0000 9227 2257, Division of Bioscience, Graduate School of Science and Technology, , Nara Institute of Science and Technology, ; 8916-5 Takayama, Ikoma, Nara 630-0192 Japan
                [2 ]GRID grid.267849.6, ISNI 0000 0001 2105 6888, Institute of Biotechnology, , Vietnam Academy of Science and Technology, ; 18 Hoang Quoc Viet road, Cau Giay, Ha Noi, Vietnam
                [3 ]GRID grid.266453.0, ISNI 0000 0001 0724 9317, Graduate School of Life Science, , University of Hyogo, ; 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297 Japan
                Article
                83890
                10.1038/s41598-021-83890-x
                7904763
                749476c2-b5ac-4c23-82d3-bd152d740d55
                © The Author(s) 2021

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: Ohsumi Frontier Science Foundation
                Funded by: FundRef http://dx.doi.org/10.13039/100010157, Noda Institute for Scientific Research;
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                © The Author(s) 2021

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                cell biology, molecular biology

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