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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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BiP binding and dissociation from deletion mutants of Ire1. The Δire1 strain KMY1516 was transformed with a mixture of linearized pRS423-IRE1-HA-HpaI and a partial fragment of the IRE1 gene carrying the desired deletion generated by overlap PCR. The transformants carrying successfully gap-repaired plasmids (deletion mutant versions of pRS423-IRE1-HA, which is a 2-μm plasmid used for expression of Ire1-HA) were selected and cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. The culturing temperature was 27°C for lanes 73–84 or 30°C for other lanes. For the empty vector (Δire1) and wild-type Ire1 (WT) controls, cells were respectively transformed with nonlinearized pRS423 and pPR423-IRE1-HA. Anti-HA immunoprecipitation was performed as described in Materials and methods, and the cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA (α-HA) and anti-BiP (α-BiP) Western blotting (WB).
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fig3: BiP binding and dissociation from deletion mutants of Ire1. The Δire1 strain KMY1516 was transformed with a mixture of linearized pRS423-IRE1-HA-HpaI and a partial fragment of the IRE1 gene carrying the desired deletion generated by overlap PCR. The transformants carrying successfully gap-repaired plasmids (deletion mutant versions of pRS423-IRE1-HA, which is a 2-μm plasmid used for expression of Ire1-HA) were selected and cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. The culturing temperature was 27°C for lanes 73–84 or 30°C for other lanes. For the empty vector (Δire1) and wild-type Ire1 (WT) controls, cells were respectively transformed with nonlinearized pRS423 and pPR423-IRE1-HA. Anti-HA immunoprecipitation was performed as described in Materials and methods, and the cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA (α-HA) and anti-BiP (α-BiP) Western blotting (WB).

Mentions: To demonstrate BiP binding to Ire1 in vivo, COOH-terminal HA-tagged Ire1 and its mutants were expressed from 2 μm plasmids, and their interaction with BiP was analyzed by coimmunoprecipitation. As described in Okamura et al. (2000), our 2-μm plasmid expression of HA-tagged Ire1 preserved ER stress inducibility and did not result in constitutive activation of the UPR pathway. Indeed, Northern blot analysis shows that in cells carrying the wild-type version of pRS423-IRE1-HA, high (80%) and very weak (< 5%) cleavage of HAC1 mRNA occurred after 60-min incubation of cells with and without 2 μg/ml tunicamycin, respectively. In the experiment shown in Fig. 3, cells were lysed under nondenaturing conditions before anti-HA immunoprecipitation, and the resulting lysates and immunoprecipitates were analyzed by anti-HA or anti-BiP Western blotting. 25 out of the 49 deletion scanning mutants were analyzed in this experiment.


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)

BiP binding and dissociation from deletion mutants of Ire1. The Δire1 strain KMY1516 was transformed with a mixture of linearized pRS423-IRE1-HA-HpaI and a partial fragment of the IRE1 gene carrying the desired deletion generated by overlap PCR. The transformants carrying successfully gap-repaired plasmids (deletion mutant versions of pRS423-IRE1-HA, which is a 2-μm plasmid used for expression of Ire1-HA) were selected and cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. The culturing temperature was 27°C for lanes 73–84 or 30°C for other lanes. For the empty vector (Δire1) and wild-type Ire1 (WT) controls, cells were respectively transformed with nonlinearized pRS423 and pPR423-IRE1-HA. Anti-HA immunoprecipitation was performed as described in Materials and methods, and the cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA (α-HA) and anti-BiP (α-BiP) Western blotting (WB).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172501&req=5

fig3: BiP binding and dissociation from deletion mutants of Ire1. The Δire1 strain KMY1516 was transformed with a mixture of linearized pRS423-IRE1-HA-HpaI and a partial fragment of the IRE1 gene carrying the desired deletion generated by overlap PCR. The transformants carrying successfully gap-repaired plasmids (deletion mutant versions of pRS423-IRE1-HA, which is a 2-μm plasmid used for expression of Ire1-HA) were selected and cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. The culturing temperature was 27°C for lanes 73–84 or 30°C for other lanes. For the empty vector (Δire1) and wild-type Ire1 (WT) controls, cells were respectively transformed with nonlinearized pRS423 and pPR423-IRE1-HA. Anti-HA immunoprecipitation was performed as described in Materials and methods, and the cell lysates (equivalent to 3 × 106 cells) and immunoprecipitates (equivalent to 1 × 107 cells) were analyzed by anti-HA (α-HA) and anti-BiP (α-BiP) Western blotting (WB).
Mentions: To demonstrate BiP binding to Ire1 in vivo, COOH-terminal HA-tagged Ire1 and its mutants were expressed from 2 μm plasmids, and their interaction with BiP was analyzed by coimmunoprecipitation. As described in Okamura et al. (2000), our 2-μm plasmid expression of HA-tagged Ire1 preserved ER stress inducibility and did not result in constitutive activation of the UPR pathway. Indeed, Northern blot analysis shows that in cells carrying the wild-type version of pRS423-IRE1-HA, high (80%) and very weak (< 5%) cleavage of HAC1 mRNA occurred after 60-min incubation of cells with and without 2 μg/ml tunicamycin, respectively. In the experiment shown in Fig. 3, cells were lysed under nondenaturing conditions before anti-HA immunoprecipitation, and the resulting lysates and immunoprecipitates were analyzed by anti-HA or anti-BiP Western blotting. 25 out of the 49 deletion scanning mutants were analyzed in this experiment.

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