Limits...
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.

Show MeSH

Related in: MedlinePlus

10-aa deletion scanning of yeast Ire1. The Δire1 strain KMY1015 harboring the UPRE-lacZ reporter plasmid pCZY1 was transformed with a mixture of linearized pPR315-IRE1-HA (a centromeric plasmid for expression of COOH-terminal HA-tagged Ire1) and a partial fragment of the IRE1 gene carrying the desired deletion. The transformants carrying the successfully gap-repaired plasmid were selected and used. For the wild-type Ire1 (WT Ire1 or WT) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. (A) Cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. Each value is the average of three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100. (B) Lysates prepared from 3 × 106 nonstressed cells using denaturing lysis buffer were analyzed by anti-HA Western blotting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172501&req=5

fig1: 10-aa deletion scanning of yeast Ire1. The Δire1 strain KMY1015 harboring the UPRE-lacZ reporter plasmid pCZY1 was transformed with a mixture of linearized pPR315-IRE1-HA (a centromeric plasmid for expression of COOH-terminal HA-tagged Ire1) and a partial fragment of the IRE1 gene carrying the desired deletion. The transformants carrying the successfully gap-repaired plasmid were selected and used. For the wild-type Ire1 (WT Ire1 or WT) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. (A) Cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. Each value is the average of three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100. (B) Lysates prepared from 3 × 106 nonstressed cells using denaturing lysis buffer were analyzed by anti-HA Western blotting.

Mentions: In Fig. 1 A, cellular UPR activity was estimated by expression of a lacZ reporter controlled by the UPRE-driven promoter (Mori et al., 1992). Cells producing the intact Ire1 protein (Fig. 1 A, WT Ire1) showed a clear UPR that was induced by tunicamycin. The tunicamycin inducibility was well preserved with mutations Δ25 to Δ95, Δ205, Δ246, Δ256, and Δ448 to Δ508, and partially with the Δ236 mutation. In contrast, little or no UPR was observed in cells carrying the mutations Δ105 to Δ195, Δ216, Δ226, or Δ266 to Δ438. As determined by Western blot detection of the COOH-terminal influenza virus HA-epitope tag, the steady-state expression of the Ire1 proteins was not significantly affected by most of the deletions that abolished the tunicamycin inducibility (Fig. 1 B), suggesting that these deletions diminished function rather than stability of Ire1. It should be noted that none of the scanning mutants rendered Ire1 constitutively active.


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)

10-aa deletion scanning of yeast Ire1. The Δire1 strain KMY1015 harboring the UPRE-lacZ reporter plasmid pCZY1 was transformed with a mixture of linearized pPR315-IRE1-HA (a centromeric plasmid for expression of COOH-terminal HA-tagged Ire1) and a partial fragment of the IRE1 gene carrying the desired deletion. The transformants carrying the successfully gap-repaired plasmid were selected and used. For the wild-type Ire1 (WT Ire1 or WT) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. (A) Cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. Each value is the average of three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100. (B) Lysates prepared from 3 × 106 nonstressed cells using denaturing lysis buffer were analyzed by anti-HA Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 10-aa deletion scanning of yeast Ire1. The Δire1 strain KMY1015 harboring the UPRE-lacZ reporter plasmid pCZY1 was transformed with a mixture of linearized pPR315-IRE1-HA (a centromeric plasmid for expression of COOH-terminal HA-tagged Ire1) and a partial fragment of the IRE1 gene carrying the desired deletion. The transformants carrying the successfully gap-repaired plasmid were selected and used. For the wild-type Ire1 (WT Ire1 or WT) and empty vector (Δire1) controls, cells were respectively transformed with nonlinearized pRS315-IRE1-HA and pPR315. (A) Cells were cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 240 min and subjected to assays for cellular β-galactosidase activity. Each value is the average of three independent transformants and was normalized to the TM+ wild-type Ire1 control, which was set at 100. (B) Lysates prepared from 3 × 106 nonstressed cells using denaturing lysis buffer were analyzed by anti-HA Western blotting.
Mentions: In Fig. 1 A, cellular UPR activity was estimated by expression of a lacZ reporter controlled by the UPRE-driven promoter (Mori et al., 1992). Cells producing the intact Ire1 protein (Fig. 1 A, WT Ire1) showed a clear UPR that was induced by tunicamycin. The tunicamycin inducibility was well preserved with mutations Δ25 to Δ95, Δ205, Δ246, Δ256, and Δ448 to Δ508, and partially with the Δ236 mutation. In contrast, little or no UPR was observed in cells carrying the mutations Δ105 to Δ195, Δ216, Δ226, or Δ266 to Δ438. As determined by Western blot detection of the COOH-terminal influenza virus HA-epitope tag, the steady-state expression of the Ire1 proteins was not significantly affected by most of the deletions that abolished the tunicamycin inducibility (Fig. 1 B), suggesting that these deletions diminished function rather than stability of Ire1. It should be noted that none of the scanning mutants rendered Ire1 constitutively active.

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