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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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Dimer formation of deletion mutants of Ire1. The Δire1 strains KMY1015 (MATα) and KMY1520 (MATa) respectively carrying deletion mutant versions of pRS315-IRE1-HA and pRS426-IRE1-FLAG, which is a 2-μm plasmid used for expression of Ire1-FLAG, were crossed to obtain diploid cells. For the empty vector control (Δire1), cells carrying pRS315 were used. The resulting diploid cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min, 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 (α-HA) and anti-FLAG (α-FLAG) Western blotting (WB; ECL signals were detected by LAS-1000plus with exposure time of 20 s to 1 min).
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fig5: Dimer formation of deletion mutants of Ire1. The Δire1 strains KMY1015 (MATα) and KMY1520 (MATa) respectively carrying deletion mutant versions of pRS315-IRE1-HA and pRS426-IRE1-FLAG, which is a 2-μm plasmid used for expression of Ire1-FLAG, were crossed to obtain diploid cells. For the empty vector control (Δire1), cells carrying pRS315 were used. The resulting diploid cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min, 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 (α-HA) and anti-FLAG (α-FLAG) Western blotting (WB; ECL signals were detected by LAS-1000plus with exposure time of 20 s to 1 min).

Mentions: Another interpretation for impaired dissociation of BiP from the Δ145 and the Δ226 mutants is that the protein folding of Ire1 was globally perturbed by these mutations, which caused targeting of BiP to the Ire1 mutants by a rather nonspecific mechanism. The following observations in Figs. 5 and 6 suggest that this interpretation is unlikely.


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)

Dimer formation of deletion mutants of Ire1. The Δire1 strains KMY1015 (MATα) and KMY1520 (MATa) respectively carrying deletion mutant versions of pRS315-IRE1-HA and pRS426-IRE1-FLAG, which is a 2-μm plasmid used for expression of Ire1-FLAG, were crossed to obtain diploid cells. For the empty vector control (Δire1), cells carrying pRS315 were used. The resulting diploid cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min, 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 (α-HA) and anti-FLAG (α-FLAG) Western blotting (WB; ECL signals were detected by LAS-1000plus with exposure time of 20 s to 1 min).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Dimer formation of deletion mutants of Ire1. The Δire1 strains KMY1015 (MATα) and KMY1520 (MATa) respectively carrying deletion mutant versions of pRS315-IRE1-HA and pRS426-IRE1-FLAG, which is a 2-μm plasmid used for expression of Ire1-FLAG, were crossed to obtain diploid cells. For the empty vector control (Δire1), cells carrying pRS315 were used. The resulting diploid cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min, 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 (α-HA) and anti-FLAG (α-FLAG) Western blotting (WB; ECL signals were detected by LAS-1000plus with exposure time of 20 s to 1 min).
Mentions: Another interpretation for impaired dissociation of BiP from the Δ145 and the Δ226 mutants is that the protein folding of Ire1 was globally perturbed by these mutations, which caused targeting of BiP to the Ire1 mutants by a rather nonspecific mechanism. The following observations in Figs. 5 and 6 suggest that this interpretation is unlikely.

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