Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins

Abstract

In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2.

Figure 1.

Schematic representation of the Kar2 protein and kar2 mutant alleles used in this study. Based on the sites of mutation, kar2 mutant alleles were categorized either as ATPase domain mutants (type A) or substrate-binding domain mutants (type S).

Figure 2.

Altered UPR in kar2 mutant strains. (A) Cells transformed with the UPRE-lacZ reporter plasmid pCZY1 were exponentially grown to an OD₆₀₀ of 0.3 at 23°C in SC medium, and at time (t = 0), the cultures were shifted to 37°C. Tunicamycin was added to a final concentration of 0 μg/ml (TM -)or2 μg/ml (TM +) at 3 min, and the cultures were subjected to a β-galactosidase assay at 200 min. Data presented are the averages of three independent transformants with SD as indicated by error bars. (B) Cells transformed with pCZY1 were exponentially grown to an OD₆₀₀ of 0.2 at 23°C in SD medium, and at time (t = 0), the cultures were shifted to 37°C (indicated as 37°C) or further cultured at 23°C (indicated as 23°C). Tunicamycin was added to a final concentration of 0 μg/ml (TM -)or 2 μg/ml (TM +) at 3 min, and the cultures were subjected to β-galactosidase assay at 200 min. Data are presented as described in the legend to the A. (C) Cells were exponentially grown to an OD₆₀₀ of 0.4 at 23°C in YPD medium, and at time (t = 0), the cultures were shifted to 37°C. Tunicamycin was added to a final concentration of 0 μg/ml (TM -)or 2 μg/ml (TM +) at 3 min, and the cells were collected at 60 min to extract total cellular RNA for Northern blot analysis using the HAC1 DNA probe. (D) Percentages of HAC1 mRNA cleavage in C were calculated as described in MATERIAL AND METHODS.

Figure 3.

Altered association or dissociation of Kar2 with Ire1 in kar2 mutant strains. The indicated strains transformed with the Ire1-HA expression plasmid pRS426-Ire1HA (lanes 2–15), or MS10 cells containing an empty vector pRS426 (lane 1) were exponentially grown to an OD₆₀₀ of 0.4 at 23°C in SC medium. At time (t = 0), the cultures were shifted to 37°C. Tunicamycin was added to a final concentration of 0 μg/ml (TM -) or 2 μg/ml (TM +) at 3 min, and cells were collected at 60 min to prepare their lysates (In lane 1, no tunicamycin was added). Anti-HA immunoprecipitates equivalent to 1 × 10⁸ cells for the lowest panel, and lysates or anti-HA immunoprecipitates equivalent to 1 × 10⁷ cells for the other panels were fractionated by 8% SDS-PAGE. Proteins were detected by subsequent Western blotting with the indicated antibodies. After treatment with ECL chemiluminescence reagents, the blots were exposed to x-ray films for the following times: 10 s for the lowest panel and 2 s for the other panels.

Figure 4.

Impaired coaggregation of Kar2 with a model unfolded protein in kar2 type S mutant strains. Cells transformed with the Δpro expression plasmid pYPR3841U or an empty vector pRS426 were exponentially grown to an OD₆₀₀ of 0.2 at 23°C in SCG medium, and the cultures were shifted to 37°C (or 34°C for the kar2-133 strain) for 60 min. Cell lysates were prepared from the resulting cultures and ultracentrifuged at 100,000 × g for 15 min, and the supernatant and pellet fractions were collected (A and B). (A) Supernatant (S) and pellet (P) fractions (equivalent to 1 × 10⁷ cells) prepared from cells containing pYPR3841U were analyzed by anti-RNAP1 Western blotting. (B) Supernatant (S; equivalent to 1 × 10⁷ cells) and pellet (P; equivalent to 5 × 10⁷ cells) fractions prepared from cells containing pYPR3841U (Δpro +) or pRS426 (Δpro -) were analyzed by anti-Kar2 Western blotting. The asterisk indicates a nonspecific band. In both panels A and B, the blots treated with ECL reagent were exposed to x-ray films for 2 s. (C) Lysates from W303 cells carrying pYPR3841U were treated with control DMSO or 2 mM DSP and ultracentrifuged. The resulting pellet fraction (input) were dissolved in 1% SDS, and subjected to immunoprecipitation with anti-Kar2 antiserum or control preimmune serum. The inputs (equivalent to 1 × 10⁷ cells for the upper panel or 3 × 10⁷ cells for the lower panel) or immunoprecipitates (equivalent to 3 × 10⁷ cells for the upper panel or 9 × 10⁷ cells for the lower panel) were analyzed by reducing SDS-PAGE followed by anti-Kar2 and anti-RNAP Western blotting.

Figure 5.

Induction of the KAR2 gene in kar2 mutants results partially from activation of the heat shock response pathway. (A) Cells were exponentially grown to an OD₆₀₀ of 0.4 at 23°C in YPD medium, and shifted to 37°C (37°C) or further cultured at 23°C (23°C) for 60 min. The resulting cells were used to extract total cellular RNA for Northern blot analysis using the KAR2, SSA1, and ACT1 DNA probes. (B) The RNAs shown in A were quantitated and normalized to ACT1 mRNA. Normalized mRNA values are presented relative to their levels of expression in MS10 cells cultured at 23°C. (C) Cells transformed with the HSE-lacZ reporter plasmid pKAR2HSE-lacZ were exponentially grown to an OD600 of 0.3 at 23°C in SC medium and shifted to 37°C for 200 min, followed by β-galactosidase assay. For an unknown reason, this HSE-lacZ reporter did not work in the W303 background strains.

Figure 6.

A schematic model to interpret our present observations. See text for explanation.