In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.
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 ...
Abstract.A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential ( ⌬⌿ m), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of ⌬⌿ m and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in ⌬⌿ m, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.
Organic field‐effect transistors (FETs) based on epitaxially grown crystals of a thiophene/phenylene co‐oligomer (see Figure and cover) are described. The FETs exhibit good operation characteristics, and the epitaxial needle‐like crystals display good charge transport properties along the needle axis. The maximum hole mobility of 0.66 cm2 V–1 s–1 is close to that of vapor phase grown oligothiophene single crystals.
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