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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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Analysis of a double mutation of kar2-113 and Ire1 subregion-V deletion. (A) The kar2-113 mutation was introduced into the Δire1 strain KMY1516 carrying either pRS313-IRE1 (untagged), pRS423-IRE1-HA (WT), or its ΔV mutant version, as described in the online supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200405153/DC1). The resulting cells and the control KAR2 cells (WT) were cultured at 23°C without extrinsic ER stress and subjected to anti-HA immunoprecipitation as described in Materials and methods. The cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA and anti-BiP Western blotting. (B) The kar2-113 mutation was introduced into KMY1516 cells carrying pRS313-IRE1 (WT) or its ΔV mutant version, as described in the online supplemental Materials and methods. The resulting cells and the control KAR2 cells (WT) were cultured at 23°C or shifted to 30°C for 120 min without extrinsic ER stress and analyzed by Northern blotting of 1 μg of total RNA using the HAC1 gene as probe. The positions of the HAC1 mRNA variants are indicated as in Fig. 7 B. The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003). (C) After spreading the cultures used in B (those at 23°C), SD agar plates were incubated at 33°C for 2 d and colonies were photographed.
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fig8: Analysis of a double mutation of kar2-113 and Ire1 subregion-V deletion. (A) The kar2-113 mutation was introduced into the Δire1 strain KMY1516 carrying either pRS313-IRE1 (untagged), pRS423-IRE1-HA (WT), or its ΔV mutant version, as described in the online supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200405153/DC1). The resulting cells and the control KAR2 cells (WT) were cultured at 23°C without extrinsic ER stress and subjected to anti-HA immunoprecipitation as described in Materials and methods. The cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA and anti-BiP Western blotting. (B) The kar2-113 mutation was introduced into KMY1516 cells carrying pRS313-IRE1 (WT) or its ΔV mutant version, as described in the online supplemental Materials and methods. The resulting cells and the control KAR2 cells (WT) were cultured at 23°C or shifted to 30°C for 120 min without extrinsic ER stress and analyzed by Northern blotting of 1 μg of total RNA using the HAC1 gene as probe. The positions of the HAC1 mRNA variants are indicated as in Fig. 7 B. The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003). (C) After spreading the cultures used in B (those at 23°C), SD agar plates were incubated at 33°C for 2 d and colonies were photographed.

Mentions: We previously reported that in the temperature-sensitive mutant alleles of the KAR2 gene encoding BiP with single amino acid changes in the ATPase domain, and BiP dissociation from Ire1 and activation of the UPR pathway are impaired when cells are cultured at a restrictive temperature (Kimata et al., 2003). One prediction from the present work is that deletion of Ire1 subregion V, abolishing BiP binding to Ire1, may suppress the UPR repression phenotype of the kar2 mutants. To test this possibility, we introduced the kar2-113 mutation, a commonly used mutant allele of the BiP ATPase domain, into wild-type and ΔV Ire1 strains. As shown in Fig. 8 A, binding of BiP to Ire1 was abolished by the Ire1 ΔV mutation not only in KAR2 but also in kar2-113 cells.


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)

Analysis of a double mutation of kar2-113 and Ire1 subregion-V deletion. (A) The kar2-113 mutation was introduced into the Δire1 strain KMY1516 carrying either pRS313-IRE1 (untagged), pRS423-IRE1-HA (WT), or its ΔV mutant version, as described in the online supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200405153/DC1). The resulting cells and the control KAR2 cells (WT) were cultured at 23°C without extrinsic ER stress and subjected to anti-HA immunoprecipitation as described in Materials and methods. The cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA and anti-BiP Western blotting. (B) The kar2-113 mutation was introduced into KMY1516 cells carrying pRS313-IRE1 (WT) or its ΔV mutant version, as described in the online supplemental Materials and methods. The resulting cells and the control KAR2 cells (WT) were cultured at 23°C or shifted to 30°C for 120 min without extrinsic ER stress and analyzed by Northern blotting of 1 μg of total RNA using the HAC1 gene as probe. The positions of the HAC1 mRNA variants are indicated as in Fig. 7 B. The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003). (C) After spreading the cultures used in B (those at 23°C), SD agar plates were incubated at 33°C for 2 d and colonies were photographed.
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fig8: Analysis of a double mutation of kar2-113 and Ire1 subregion-V deletion. (A) The kar2-113 mutation was introduced into the Δire1 strain KMY1516 carrying either pRS313-IRE1 (untagged), pRS423-IRE1-HA (WT), or its ΔV mutant version, as described in the online supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200405153/DC1). The resulting cells and the control KAR2 cells (WT) were cultured at 23°C without extrinsic ER stress and subjected to anti-HA immunoprecipitation as described in Materials and methods. The cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA and anti-BiP Western blotting. (B) The kar2-113 mutation was introduced into KMY1516 cells carrying pRS313-IRE1 (WT) or its ΔV mutant version, as described in the online supplemental Materials and methods. The resulting cells and the control KAR2 cells (WT) were cultured at 23°C or shifted to 30°C for 120 min without extrinsic ER stress and analyzed by Northern blotting of 1 μg of total RNA using the HAC1 gene as probe. The positions of the HAC1 mRNA variants are indicated as in Fig. 7 B. The percentage of HAC1 mRNA cleavage was estimated as described in Kimata et al. (2003). (C) After spreading the cultures used in B (those at 23°C), SD agar plates were incubated at 33°C for 2 d and colonies were photographed.
Mentions: We previously reported that in the temperature-sensitive mutant alleles of the KAR2 gene encoding BiP with single amino acid changes in the ATPase domain, and BiP dissociation from Ire1 and activation of the UPR pathway are impaired when cells are cultured at a restrictive temperature (Kimata et al., 2003). One prediction from the present work is that deletion of Ire1 subregion V, abolishing BiP binding to Ire1, may suppress the UPR repression phenotype of the kar2 mutants. To test this possibility, we introduced the kar2-113 mutation, a commonly used mutant allele of the BiP ATPase domain, into wild-type and ΔV Ire1 strains. As shown in Fig. 8 A, binding of BiP to Ire1 was abolished by the Ire1 ΔV mutation not only in KAR2 but also in kar2-113 cells.

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