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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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Activity of Ire1 luminal-domain mutants other than those used in the 10-aa deletion scanning. (A) The insertion and deletions carried on the mutants. III-G15 is a (Gly)15 spacer insertion in subregion III, and Δ448−483, Δ482−517, and ΔV are wide-range deletions in subregion V. (B) KMY1015 (Δire1) cells carrying both pCZY1 (UPRE-lacZ reporter) and a mutant version of pRS315-IRE1-HA were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. For the wild-type Ire1 (WT Ire1) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. Each value is the average from three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100.
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fig2: Activity of Ire1 luminal-domain mutants other than those used in the 10-aa deletion scanning. (A) The insertion and deletions carried on the mutants. III-G15 is a (Gly)15 spacer insertion in subregion III, and Δ448−483, Δ482−517, and ΔV are wide-range deletions in subregion V. (B) KMY1015 (Δire1) cells carrying both pCZY1 (UPRE-lacZ reporter) and a mutant version of pRS315-IRE1-HA were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. For the wild-type Ire1 (WT Ire1) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. Each value is the average from three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100.

Mentions: An insertion mutant shown in Fig. 2 A supports our prediction that the “functionally indispensable” region is structurally divided into subregion II and IV. As described in the online supplemental Materials and methods, we generated an Ire1 mutant with a (Gly)15 spacer insertion in subregion III (named III-G15), and found this mutant functioned almost as well as wild type to activate the UPR pathway (Fig. 2 B).


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)

Activity of Ire1 luminal-domain mutants other than those used in the 10-aa deletion scanning. (A) The insertion and deletions carried on the mutants. III-G15 is a (Gly)15 spacer insertion in subregion III, and Δ448−483, Δ482−517, and ΔV are wide-range deletions in subregion V. (B) KMY1015 (Δire1) cells carrying both pCZY1 (UPRE-lacZ reporter) and a mutant version of pRS315-IRE1-HA were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. For the wild-type Ire1 (WT Ire1) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. Each value is the average from three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Activity of Ire1 luminal-domain mutants other than those used in the 10-aa deletion scanning. (A) The insertion and deletions carried on the mutants. III-G15 is a (Gly)15 spacer insertion in subregion III, and Δ448−483, Δ482−517, and ΔV are wide-range deletions in subregion V. (B) KMY1015 (Δire1) cells carrying both pCZY1 (UPRE-lacZ reporter) and a mutant version of pRS315-IRE1-HA were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. For the wild-type Ire1 (WT Ire1) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. Each value is the average from three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100.
Mentions: An insertion mutant shown in Fig. 2 A supports our prediction that the “functionally indispensable” region is structurally divided into subregion II and IV. As described in the online supplemental Materials and methods, we generated an Ire1 mutant with a (Gly)15 spacer insertion in subregion III (named III-G15), and found this mutant functioned almost as well as wild type to activate the UPR pathway (Fig. 2 B).

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