Abstract
The unfolded protein response (UPR) is crucial to life by regulating the cellular response to the stress in the endoplasmic reticulum (ER) imposed by abiotic and biotic cues such as heat shock and viral infection. The inositol requiring enzyme 1 (IRE1) signaling pathway activated by the IRE1-mediated unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans, is the most ancient branch of the UPR. In this study, we systematically examined yeast IRE1p-HAC1, plant IRE1A/IRE1B-bZIP60 and human hIRE1-XBP1 pairs. We found that, unlike bZIP60, XBP1 is unable to functionally swap HAC1p in yeast, and that the inter-species heterotypic interactions among HAC1p, bZIP60 and XBP1 are not permitted. These data demonstrate evolutionary divergence of the downstream signaling of IRE1-bZIP. We also discovered that the dual cytosolic domains of plant IRE1s act in vivo in a mechanism consistent with IRE1p and hIRE1, and that plant IRE1B not only interacts with IRE1p but also forms typical IRE1 dynamic foci in yeast. Thus, the upstream components of the IRE1 signaling branch including IRE1 activation and action mechanisms are highly conserved. Taken together these data advance the molecular understanding of evolutionary divergence and conservation of the IRE1 signaling pathway across kingdoms.
Highlights
Upon translation, newly synthesized proteins are loaded in an unfolded state into the lumen of the endoplasmic reticulum (ER), where they undergo folding and posttranslational modifications aided by ER-resident chaperones to reach maturity[1,2,3]
Like HAC1 and XBP1 mRNAs, the un-spliced bZIP60 mRNA precursor can fold into an inositol requiring enzyme 1 (IRE1) recognition site composed of two stem loops, each possessing the bases at three positions strictly conserved from yeast to mammalians[14,15,24,25,26]
To further examine whether this yeast gene could be functionally replaced by XBP1, the human mRNA part of the IRE1-mediated unfolded protein response (UPR) arm[5,6], we designed three types of XBP1, i.e., XBP1 U, XBP1 S and XBP1 Si (Supplementary Fig. S1). cDNAs of HAC1p U, HAC1p S and XBP1 variants were expressed in a HAC1-deficient yeast strain (CRY1 Δhac1p::TRP)[45,46] using a CEN-ARS plasmid containing a GAL1 inducible promoter
Summary
Newly synthesized proteins are loaded in an unfolded state into the lumen of the endoplasmic reticulum (ER), where they undergo folding and posttranslational modifications aided by ER-resident chaperones to reach maturity[1,2,3]. The unique output of the IRE1 signaling is the RNase-mediated site-specific cleavage of an mRNA, the product of HAC1 in yeast[11], XBP1 in metazoan[12,13], and bZIP60 in plants[14,15]. Like HAC1 and XBP1 mRNAs, the un-spliced bZIP60 (bZIP60 U) mRNA precursor can fold into an IRE1 recognition site composed of two stem loops, each possessing the bases at three positions strictly conserved from yeast to mammalians[14,15,24,25,26]. Upon ER stress, oligomeric assembly of the ER-luminal domain induces IRE1p clustering in the ER membrane, facilitating the formation of the discrete foci of higher-order oligomers and the docking of HAC1 U mRNA onto a positively charged motif that is in proximity to the kinase/RNase and transmembrane domains[31,32,33,34]. The precisely controlled molecular process leading to the unconventional splicing has been posited to contribute to efficiency and selectivity and, fidelity in UPR signaling[31,32,34]
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