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First published online April 23, 2007
doi: 10.1242/10.1242/jcs.002808

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Self-association and BiP dissociation are not sufficient for activation of the ER stress sensor Ire1

Laboratory of Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0192, Japan

View larger version (31K): [in this window] [in a new window]   Fig. 1. Ire1 mutants and their cellular expression. (A) The luminal domain of wild-type (WT) Ire1 and luminal domain mutants is shown schematically. Ire1 is inactivated by internal 10-a.a. deletions in the shaded region, and subregions I to V were designated based on this observation (Kimata et al., 2004) (subregion I corresponds to a.a. positions 32-111; subregion II, 112-242; subregion III, 243-272; subregion IV, 273-454; and subregion V, 455-524). The I and the V mutations are deletions of T32 to R91 and T463 to N523, respectively. The core mutation is the combination of the I and V mutations. The position of the S103P point mutation is also indicated. (B) Lysates were prepared from KMY1015 (ire1 null mutant) cells carrying pRS315-Ire1-HA (centromeric plasmid for expression of Ire1-HA) or mutant alleles under denaturing conditions, and subjected to anti-HA western blotting with 15 µg total protein per lane. The empty vector control (ire1) carried pRS315 (Sikorski and Hieter, 1989) instead of pRS315-Ire1-HA.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Activity of the Ire1 mutants as measured by a UPRE-lacZ reporter. KMY1015 (ire1 null mutant) cells carrying both pCZY1 (UPRE-lacZ reporter plasmid) and pRS315-Ire1-HA or mutant alleles were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 4 hours and subjected to -galactosidase assay. For the empty vector control (ire1), pRS315 was used instead of pRS315-Ire1-HA. In all three panels, each value is the mean ± s.d. for three independent clones and was normalized to that of the Tun+ wild-type Ire1-HA control, which was set at 100.

View larger version (26K): [in this window] [in a new window]   Fig. 3. HAC1u cleavage by the Ire1 luminal domain mutants. KMY1516 (ire1 null mutant) cells carrying pRS313-Ire1-HA (centromeric plasmid for expression of Ire1-HA) or mutant alleles were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes and total RNA was analyzed by northern blotting using the HAC1 probe. For the empty vector control (ire1), pRS313 was used instead of pRS313-Ire1-HA. The positions of the uncleaved (HAC1u), the cleaved and ligated (HAC1i) and the cleaved but unligated (5'-cleaved and 3'-cleaved) versions of HAC1 mRNA are indicated (Kawahara et al., 1998). The percentage of HAC1 mRNA cleavage was calculated as described in the Materials and Methods.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Binding of BiP to luminal domain mutated Ire1. KMY1516 (ire1 null mutant) cells carrying pRS423-Ire1-HA (2-µm plasmid for expression of Ire1-HA) or mutant alleles were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes and their lysates were then used for anti-HA immunoprecipitation. The cell lysates (equivalent to 3x106 cells) and the anti-HA immunoprecipitates (equivalent to 1x107 cells; -HA IP) were analyzed by anti-HA (-HA) and anti-BiP (-BiP) western blotting (WB). For the empty vector control (ire1), pRS423 was used instead of pRS423-Ire1-HA.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Self-association of the Ire1 luminal domain mutants. (A) KMY1015 (ire1 null mutant) cells carrying both pRS426-Ire1-Flag (2-µm plasmid for expression of Ire1-Flag; wild type (WT) or mutant) and pRS315-Ire1-HA (WT or mutant) were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes, and lysates were then used for anti-HA immunoprecipitation. The cell lysates (equivalent to 3x106 cells) and the anti-HA immunoprecipitates (equivalent to 1x107 cells; -HA IP) were analyzed by anti-HA (-HA) and anti-Flag (-Flag) western blotting (WB). The ratios of Ire1-Flag signal to Ire1-HA signal in -HA IP were normalized to the Tun+ WT Ire1 control, which was set at 1.0, and are indicated. (B) KMY1015 cells carrying pRS315-Ire1-HA (core or S103P core mutant) were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes, and their lysates were fractionated by 5-25% glycerol gradient centrifugation. Ire1-HA in each fraction was detected by anti-HA immunoprecipitation followed by anti-HA Western blotting. The positions of protein Mr markers, fractionated in parallel on an identical gradient, are indicated.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Analysis of additional amino acid substitutions at and around S103 in Ire1. (A) Amino acid sequences of the mutant alleles used in this assay. The mutated residues are in bold. Note that all alleles carry the core mutation. (B,C) KMY1015 (ire1 null mutant) cells carrying both the UPRE-lacZ reporter pCZY1 and a mutant allele of pRS315-Ire1-HA were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 4 hours, and subjected to -galactosidase assay. Each value is the mean ± s.d. for three independent clones and was normalized to that of the Tun+ wild-type Ire1-HA control, which was set at 100. (D) KMY1015 (ire1 null mutant) cells carrying the indicated mutant alleles of pRS315-Ire1-HA were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes, and total RNA was analyzed by northern blotting using the HAC1 probe. See Fig. 3 legend for further details.