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A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1.

Kimata Y, Oikawa D, Shimizu Y, Ishiwata-Kimata Y, Kohno K - J. Cell Biol. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. kimata@bs.naist.jp

ABSTRACT
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.

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Hypersensitive response of Ire1 with a subregion-V deletion not to conventional ER stressors, but to high temperature and ethanol. (A) The Δire1 strain KMY1015 carrying either empty vector pRS315 (Δire1), pRS315-IRE1-HA (WT), or its ΔV mutant version was cultured under nonstressed conditions, and its lysate (equivalent to 3 × 106 cells; prepared using denaturing lysis buffer) was analyzed by anti-HA Western blotting. (B) A Δire1 strain KMY1516 carrying either pRS313-IRE1 (a centromeric plasmid for expression of untagged Ire1; WT) or its ΔV mutant version was cultured under nonstressed conditions of 30°C, treated with tunicamycin (TM) at 2 μg/ml for the indicated time (top and second panels) or at the indicated concentration for 60 min (third panel), DTT at the indicated concentration for 40 min (fourth panel) or 8% ethanol for the indicated time (fifth panel), or shifted to 37°C for 30 min and then to 39°C for 30 min (fifth panel, Temp. shift). For the bottom panel, cells treated with 2 μg/ml tunicamycin for 60 min were washed twice with fresh SD medium and further cultured for the indicated time. 1 μg of total RNA prepared from these cells was analyzed by Northern blotting using the HAC1 gene as probe. The positions of uncleaved (HAC1u), cleaved then ligated (HAC1i), and cleaved but unligated (5′-cleaved and 3′-cleaved) versions of HAC1 mRNA are indicated (Kawahara et al., 1998). The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003).
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fig7: Hypersensitive response of Ire1 with a subregion-V deletion not to conventional ER stressors, but to high temperature and ethanol. (A) The Δire1 strain KMY1015 carrying either empty vector pRS315 (Δire1), pRS315-IRE1-HA (WT), or its ΔV mutant version was cultured under nonstressed conditions, and its lysate (equivalent to 3 × 106 cells; prepared using denaturing lysis buffer) was analyzed by anti-HA Western blotting. (B) A Δire1 strain KMY1516 carrying either pRS313-IRE1 (a centromeric plasmid for expression of untagged Ire1; WT) or its ΔV mutant version was cultured under nonstressed conditions of 30°C, treated with tunicamycin (TM) at 2 μg/ml for the indicated time (top and second panels) or at the indicated concentration for 60 min (third panel), DTT at the indicated concentration for 40 min (fourth panel) or 8% ethanol for the indicated time (fifth panel), or shifted to 37°C for 30 min and then to 39°C for 30 min (fifth panel, Temp. shift). For the bottom panel, cells treated with 2 μg/ml tunicamycin for 60 min were washed twice with fresh SD medium and further cultured for the indicated time. 1 μg of total RNA prepared from these cells was analyzed by Northern blotting using the HAC1 gene as probe. The positions of uncleaved (HAC1u), cleaved then ligated (HAC1i), and cleaved but unligated (5′-cleaved and 3′-cleaved) versions of HAC1 mRNA are indicated (Kawahara et al., 1998). The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003).

Mentions: We performed a detailed phenotypic analysis of the ΔV mutant to understand what happens when the binding of BiP to Ire1 is abolished. First, Ire1 and its ΔV version were expressed as HA-tagged proteins from the centromeric plasmids and were detected by anti-HA Western blotting of the cell lysates. As shown in Fig. 7 A, the ΔV mutation caused a small (∼20%) diminution of the steady-state level of the Ire1 protein.


A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1.

Kimata Y, Oikawa D, Shimizu Y, Ishiwata-Kimata Y, Kohno K - J. Cell Biol. (2004)

Hypersensitive response of Ire1 with a subregion-V deletion not to conventional ER stressors, but to high temperature and ethanol. (A) The Δire1 strain KMY1015 carrying either empty vector pRS315 (Δire1), pRS315-IRE1-HA (WT), or its ΔV mutant version was cultured under nonstressed conditions, and its lysate (equivalent to 3 × 106 cells; prepared using denaturing lysis buffer) was analyzed by anti-HA Western blotting. (B) A Δire1 strain KMY1516 carrying either pRS313-IRE1 (a centromeric plasmid for expression of untagged Ire1; WT) or its ΔV mutant version was cultured under nonstressed conditions of 30°C, treated with tunicamycin (TM) at 2 μg/ml for the indicated time (top and second panels) or at the indicated concentration for 60 min (third panel), DTT at the indicated concentration for 40 min (fourth panel) or 8% ethanol for the indicated time (fifth panel), or shifted to 37°C for 30 min and then to 39°C for 30 min (fifth panel, Temp. shift). For the bottom panel, cells treated with 2 μg/ml tunicamycin for 60 min were washed twice with fresh SD medium and further cultured for the indicated time. 1 μg of total RNA prepared from these cells was analyzed by Northern blotting using the HAC1 gene as probe. The positions of uncleaved (HAC1u), cleaved then ligated (HAC1i), and cleaved but unligated (5′-cleaved and 3′-cleaved) versions of HAC1 mRNA are indicated (Kawahara et al., 1998). The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003).
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fig7: Hypersensitive response of Ire1 with a subregion-V deletion not to conventional ER stressors, but to high temperature and ethanol. (A) The Δire1 strain KMY1015 carrying either empty vector pRS315 (Δire1), pRS315-IRE1-HA (WT), or its ΔV mutant version was cultured under nonstressed conditions, and its lysate (equivalent to 3 × 106 cells; prepared using denaturing lysis buffer) was analyzed by anti-HA Western blotting. (B) A Δire1 strain KMY1516 carrying either pRS313-IRE1 (a centromeric plasmid for expression of untagged Ire1; WT) or its ΔV mutant version was cultured under nonstressed conditions of 30°C, treated with tunicamycin (TM) at 2 μg/ml for the indicated time (top and second panels) or at the indicated concentration for 60 min (third panel), DTT at the indicated concentration for 40 min (fourth panel) or 8% ethanol for the indicated time (fifth panel), or shifted to 37°C for 30 min and then to 39°C for 30 min (fifth panel, Temp. shift). For the bottom panel, cells treated with 2 μg/ml tunicamycin for 60 min were washed twice with fresh SD medium and further cultured for the indicated time. 1 μg of total RNA prepared from these cells was analyzed by Northern blotting using the HAC1 gene as probe. The positions of uncleaved (HAC1u), cleaved then ligated (HAC1i), and cleaved but unligated (5′-cleaved and 3′-cleaved) versions of HAC1 mRNA are indicated (Kawahara et al., 1998). The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003).
Mentions: We performed a detailed phenotypic analysis of the ΔV mutant to understand what happens when the binding of BiP to Ire1 is abolished. First, Ire1 and its ΔV version were expressed as HA-tagged proteins from the centromeric plasmids and were detected by anti-HA Western blotting of the cell lysates. As shown in Fig. 7 A, the ΔV mutation caused a small (∼20%) diminution of the steady-state level of the Ire1 protein.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. kimata@bs.naist.jp

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus