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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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Proteolytic cleavage of Ire1 to assign the BiP-binding site. (A) The III-IEGR mutation in Ire1. The Factor Xa cleavage sequence is underlined. The position of the sequence against which the anti-Ire1 NH2-terminal peptide antibody was generated is also indicated. (B) The Δire1 strain KMY1516 carrying pPR423-IRE1-HA (WT) or its III-IEGR version was cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. 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 and anti-BiP Western blotting (WB). (C–E) Proteolytic cleavage analysis. KMY1516 cells carrying the III-IEGR version of pPR423-IRE1-HA were subjected to anti-HA immunoprecipitation, and 15 μl of the resulting immunoprecipitate (equivalent to 1 × 108 cells), the constituents of which were deduced as illustrated in C, was incubated with the indicated concentration of Factor Xa as described in Materials and methods. Note that in C the BiP-binding site is set in subregion V, as concluded in this work. In D, one-tenth portions of the resulting bead-bound and released fractions were fractionated by SDS-PAGE (8%) followed by immunoblotting using the indicated antibodies, and ECL signals were detected by LAS-1000plus (exposure time of 20 s for anti-BiP, 15 s for anti-HA, and 10 s for the anti-Ire1 NH2-terminal peptide). The positions of the full-length (a), slightly truncated (b), and Factor Xa-cleaved (c) Ire1-HA are indicated. Note that the slight truncation of Ire1-HA occurred independently of Factor Xa in Factor Xa cleavage buffer. In E, anti-HA immunoprecipitate from KMY1516 cells carrying the empty vector pRS423 was used as a negative control (Δire1), and one-third portions of the released fractions were subjected to dot blotting using antibody against the anti-Ire1 NH2-terminal peptide.
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fig4: Proteolytic cleavage of Ire1 to assign the BiP-binding site. (A) The III-IEGR mutation in Ire1. The Factor Xa cleavage sequence is underlined. The position of the sequence against which the anti-Ire1 NH2-terminal peptide antibody was generated is also indicated. (B) The Δire1 strain KMY1516 carrying pPR423-IRE1-HA (WT) or its III-IEGR version was cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. 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 and anti-BiP Western blotting (WB). (C–E) Proteolytic cleavage analysis. KMY1516 cells carrying the III-IEGR version of pPR423-IRE1-HA were subjected to anti-HA immunoprecipitation, and 15 μl of the resulting immunoprecipitate (equivalent to 1 × 108 cells), the constituents of which were deduced as illustrated in C, was incubated with the indicated concentration of Factor Xa as described in Materials and methods. Note that in C the BiP-binding site is set in subregion V, as concluded in this work. In D, one-tenth portions of the resulting bead-bound and released fractions were fractionated by SDS-PAGE (8%) followed by immunoblotting using the indicated antibodies, and ECL signals were detected by LAS-1000plus (exposure time of 20 s for anti-BiP, 15 s for anti-HA, and 10 s for the anti-Ire1 NH2-terminal peptide). The positions of the full-length (a), slightly truncated (b), and Factor Xa-cleaved (c) Ire1-HA are indicated. Note that the slight truncation of Ire1-HA occurred independently of Factor Xa in Factor Xa cleavage buffer. In E, anti-HA immunoprecipitate from KMY1516 cells carrying the empty vector pRS423 was used as a negative control (Δire1), and one-third portions of the released fractions were subjected to dot blotting using antibody against the anti-Ire1 NH2-terminal peptide.

Mentions: Another approach to determine the BiP-binding site on Ire1 was restricted proteolysis of the BiP-bound Ire1 protein. In this experiment, an HA-tagged version of an Ire1 mutant carrying an insertion of the Factor Xa cleavage site (Ile-Glu-Gly-Arg) in subregion III (Ire1 III-IEGR; Fig. 4 A) was expressed in yeast cells. Tunicamycin treatment of cells and subsequent anti-HA immunoprecipitation indicated that the III-IEGR mutation did not cause a change from the wild type in BiP binding or dissociation in vivo (Fig. 4 B). Nonstressed cells producing III-IEGR Ire1 were subjected to anti-HA immunoprecipitation, and the immunoprecipitate, the constituents of which were deduced as illustrated in Fig. 4 C, was used for the following proteolytic cleavage analysis. As shown by anti-HA Western blotting (Fig. 4 D), treatment with Factor Xa diminished the full-length (Fig. 4 D, a) and slightly truncated (Fig. 4 D, b) Ire1 proteins and yielded an ∼105-kD version (Fig. 4 D, c) in the bead-bound fraction. The migration on SDS-PAGE was consistent with the deduced size of the COOH-terminal fragment produced by the Factor Xa cleavage (101 kD), and, as expected, this protein was undetectable with an Ire1 NH2-terminal antibody that clearly detects the full-length Ire1 (Fig. 4 D, WB: α-Ire1 NH2-terminal peptide). We could not detect the NH2-terminal fragment (deduced to be 25 kD) in either the bead-bound fraction or in the released fraction by the anti-Ire1 NH2-terminal Western blot analysis (unpublished data), probably because this fragment was further cleaved nonspecifically. The presence of the NH2-terminal fragment in the released fraction was indicated by the dot blot analysis shown in Fig. 4 E. The anti-BiP Western blot shown in Fig. 4 D indicates that the amount of BiP in the bead-bound fraction was not diminished by Factor Xa cleavage, except that signals of both Ire1-HA and BiP were reduced by nonspecific proteolysis at 40 μg/ml of Factor Xa. This finding strongly suggests that the BiP-binding site is in the COOH-terminal fragment. Based on this proteolysis experiment and the aforementioned analysis of the deletion mutants, we have concluded that the BiP-binding site lies in subregion V.


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)

Proteolytic cleavage of Ire1 to assign the BiP-binding site. (A) The III-IEGR mutation in Ire1. The Factor Xa cleavage sequence is underlined. The position of the sequence against which the anti-Ire1 NH2-terminal peptide antibody was generated is also indicated. (B) The Δire1 strain KMY1516 carrying pPR423-IRE1-HA (WT) or its III-IEGR version was cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. 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 and anti-BiP Western blotting (WB). (C–E) Proteolytic cleavage analysis. KMY1516 cells carrying the III-IEGR version of pPR423-IRE1-HA were subjected to anti-HA immunoprecipitation, and 15 μl of the resulting immunoprecipitate (equivalent to 1 × 108 cells), the constituents of which were deduced as illustrated in C, was incubated with the indicated concentration of Factor Xa as described in Materials and methods. Note that in C the BiP-binding site is set in subregion V, as concluded in this work. In D, one-tenth portions of the resulting bead-bound and released fractions were fractionated by SDS-PAGE (8%) followed by immunoblotting using the indicated antibodies, and ECL signals were detected by LAS-1000plus (exposure time of 20 s for anti-BiP, 15 s for anti-HA, and 10 s for the anti-Ire1 NH2-terminal peptide). The positions of the full-length (a), slightly truncated (b), and Factor Xa-cleaved (c) Ire1-HA are indicated. Note that the slight truncation of Ire1-HA occurred independently of Factor Xa in Factor Xa cleavage buffer. In E, anti-HA immunoprecipitate from KMY1516 cells carrying the empty vector pRS423 was used as a negative control (Δire1), and one-third portions of the released fractions were subjected to dot blotting using antibody against the anti-Ire1 NH2-terminal peptide.
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fig4: Proteolytic cleavage of Ire1 to assign the BiP-binding site. (A) The III-IEGR mutation in Ire1. The Factor Xa cleavage sequence is underlined. The position of the sequence against which the anti-Ire1 NH2-terminal peptide antibody was generated is also indicated. (B) The Δire1 strain KMY1516 carrying pPR423-IRE1-HA (WT) or its III-IEGR version was cultured with (+) or without (−) 2 μg/ml tunicamycin (TM) for 60 min. 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 and anti-BiP Western blotting (WB). (C–E) Proteolytic cleavage analysis. KMY1516 cells carrying the III-IEGR version of pPR423-IRE1-HA were subjected to anti-HA immunoprecipitation, and 15 μl of the resulting immunoprecipitate (equivalent to 1 × 108 cells), the constituents of which were deduced as illustrated in C, was incubated with the indicated concentration of Factor Xa as described in Materials and methods. Note that in C the BiP-binding site is set in subregion V, as concluded in this work. In D, one-tenth portions of the resulting bead-bound and released fractions were fractionated by SDS-PAGE (8%) followed by immunoblotting using the indicated antibodies, and ECL signals were detected by LAS-1000plus (exposure time of 20 s for anti-BiP, 15 s for anti-HA, and 10 s for the anti-Ire1 NH2-terminal peptide). The positions of the full-length (a), slightly truncated (b), and Factor Xa-cleaved (c) Ire1-HA are indicated. Note that the slight truncation of Ire1-HA occurred independently of Factor Xa in Factor Xa cleavage buffer. In E, anti-HA immunoprecipitate from KMY1516 cells carrying the empty vector pRS423 was used as a negative control (Δire1), and one-third portions of the released fractions were subjected to dot blotting using antibody against the anti-Ire1 NH2-terminal peptide.
Mentions: Another approach to determine the BiP-binding site on Ire1 was restricted proteolysis of the BiP-bound Ire1 protein. In this experiment, an HA-tagged version of an Ire1 mutant carrying an insertion of the Factor Xa cleavage site (Ile-Glu-Gly-Arg) in subregion III (Ire1 III-IEGR; Fig. 4 A) was expressed in yeast cells. Tunicamycin treatment of cells and subsequent anti-HA immunoprecipitation indicated that the III-IEGR mutation did not cause a change from the wild type in BiP binding or dissociation in vivo (Fig. 4 B). Nonstressed cells producing III-IEGR Ire1 were subjected to anti-HA immunoprecipitation, and the immunoprecipitate, the constituents of which were deduced as illustrated in Fig. 4 C, was used for the following proteolytic cleavage analysis. As shown by anti-HA Western blotting (Fig. 4 D), treatment with Factor Xa diminished the full-length (Fig. 4 D, a) and slightly truncated (Fig. 4 D, b) Ire1 proteins and yielded an ∼105-kD version (Fig. 4 D, c) in the bead-bound fraction. The migration on SDS-PAGE was consistent with the deduced size of the COOH-terminal fragment produced by the Factor Xa cleavage (101 kD), and, as expected, this protein was undetectable with an Ire1 NH2-terminal antibody that clearly detects the full-length Ire1 (Fig. 4 D, WB: α-Ire1 NH2-terminal peptide). We could not detect the NH2-terminal fragment (deduced to be 25 kD) in either the bead-bound fraction or in the released fraction by the anti-Ire1 NH2-terminal Western blot analysis (unpublished data), probably because this fragment was further cleaved nonspecifically. The presence of the NH2-terminal fragment in the released fraction was indicated by the dot blot analysis shown in Fig. 4 E. The anti-BiP Western blot shown in Fig. 4 D indicates that the amount of BiP in the bead-bound fraction was not diminished by Factor Xa cleavage, except that signals of both Ire1-HA and BiP were reduced by nonspecific proteolysis at 40 μg/ml of Factor Xa. This finding strongly suggests that the BiP-binding site is in the COOH-terminal fragment. Based on this proteolysis experiment and the aforementioned analysis of the deletion mutants, we have concluded that the BiP-binding site lies in subregion V.

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