Genetic Evidence for a Role of BiP/Kar2 That Regulates Ire1 in Response to Accumulation of Unfolded Proteins

Abstract

In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperaturesensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substratebinding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2. INTRODUCTIONAccumulation of unfolded proteins in the endoplasmic reticulum (ER) results in the transcriptional induction of various genes including those encoding ER-resident chaperones and folding enzymes. This cellular mechanism is called the unfolded protein response (UPR), and several important features of the UPR signaling pathway have been revealed initially through studies in the budding yeast Saccharomyces cerevisiae. Yeast Ire1 is a 1115-amino acid transmembrane protein that transmits the unfolded protein signal across the ER membrane (Cox et al., 1993;Mori et al., 1993). The cytoplasmic domain (C-terminal half) of Ire1 possesses both serine/threonine kinase and site-specific endoribonuclease activities Sidrauski and Walter, 1997). Ire1 dimerizes in response to the accumulation of unfolded proteins, resulting in its trans-autophosphorylation and activation (Shamu and Walter, 1996;Welihinda and Kaufman, 1996). Activated Ire1 in turn promotes splicing of HAC1 precursor mRNA to produce the mature form (Cox and Walter, 1996;Sidrauski and Walter, 1997), which is effectively translated into a functional transcription factor (Mori et al., 2000;Ruegsegger et al., 2001). Mature form of Hac1 efficiently induces transcription of UPR target genes containing the UPR element (UPRE) in their promoter region (Mori et al., 1992;Kohno et al., 1993).In mammalian cells, accumulation of unfolded proteins initiates signaling from the ER via more complicated pathways. Two homologues of Ire1, Ire1␣ and Ire1␤, have been identified (Tirasophon et al., 1998;Wang et al., 1998;Iwawaki et al., 2001). According to recent reports (Yoshida et al., 2001;Calfon et al., 2002), IRE1 functions to promote splicing of an mRNA encoding ...

Antifungal compound honokiol triggers oxidative stress responsive signalling pathway and modulates central carbon metabolism

“…Consistently, we found that “Atf1 activated” categories are over-represented in the statistical test (52 genes in total, p -value = 1.20E-43). We, therefore, did the combinational and comparative analysis between our results and the previous published microarray data of fission yeast responding to various environmental stresses (oxidative stress, heavy metal stress, osmotic stress, heat shock and DNA damage) (Dongrong Chen et al, 2003). As shown in Figure 2, the result of hierarchical analysis indicates that honokiol-caused transcriptomic modulation is significantly consistent with stress-caused changes, especially for H 2 O 2 , Cd and heat-induced stresses.…”

“…Honokiol-1, -2, -3 represent three biological replicates. Other stress treatment conditions are reported before (Dongrong et al 2003). Briefly, these stress conditions include: oxidative stress (0.5 mM H 2 O 2 , treat 15 and 60 min), heavy metal stress (0.5 mM CdSO 4 , treat 15 and 60 min), heat stress (39°C, treat 15 and 60 min), osmotic stress (1 M sorbitol, treat 15 and 60 min) and alkylating agent (0.2%, w/v methylmethane sulfonate, treat 15 and 60 min).…”

On the mechanism of sensing unfolded protein in the endoplasmic reticulum

“…The dissociation of ER chaperones from Ire1's luminal domain (LD) as they become engaged with unfolded proteins is widely held to be the mechanistic step that triggers Ire1 activation. Indeed, Ire1 activation is temporally linked to reversible dissociation from ER-luminal chaperones, most notably BiP (15,16). However, genetic and structural evidence in direct support of the notion that Ire1-BiP dissociation is mechanistically important for Ire1 activation, and not merely correlative, has not been readily forthcoming.…”

The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response

“…However, the UPR represents a novel intracellular ER transmembrane signaling pathway, which appears to employ a ligand-independent activation mechanism (14) in which the IRE1 N-terminal luminal domain (NLD) functions as an ER stress sensor. According to this model, under normal conditions IRE1 is maintained in a monomeric state through interaction of the NLD with the ER resident chaperone BiP (15)(16)(17)(18)(19). Upon ER stress, immunoglobin-binding protein (BiP)͞glucose-regulated protein of 78 kDa (Grp78) (BiP) binds to unfolded proteins as they accumulate, permitting the released NLD to form homodimers.…”