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. 2010 Jul 6;8(7):e1000415.
doi: 10.1371/journal.pbio.1000415.

BiP binding to the ER-stress sensor Ire1 tunes the homeostatic behavior of the unfolded protein response

David Pincus  1 , Michael W Chevalier, Tomás Aragón, Eelco van Anken, Simon E Vidal, Hana El-Samad, Peter Walter
Affiliations

BiP binding to the ER-stress sensor Ire1 tunes the homeostatic behavior of the unfolded protein response

David Pincus et al. PLoS Biol. .

Abstract

The unfolded protein response (UPR) is an intracellular signaling pathway that counteracts variable stresses that impair protein folding in the endoplasmic reticulum (ER). As such, the UPR is thought to be a homeostat that finely tunes ER protein folding capacity and ER abundance according to need. The mechanism by which the ER stress sensor Ire1 is activated by unfolded proteins and the role that the ER chaperone protein BiP plays in Ire1 regulation have remained unclear. Here we show that the UPR matches its output to the magnitude of the stress by regulating the duration of Ire1 signaling. BiP binding to Ire1 serves to desensitize Ire1 to low levels of stress and promotes its deactivation when favorable folding conditions are restored to the ER. We propose that, mechanistically, BiP achieves these functions by sequestering inactive Ire1 molecules, thereby providing a barrier to oligomerization and activation, and a stabilizing interaction that facilitates de-oligomerization and deactivation. Thus BiP binding to or release from Ire1 is not instrumental for switching the UPR on and off as previously posed. By contrast, BiP provides a buffer for inactive Ire1 molecules that ensures an appropriate response to restore protein folding homeostasis to the ER by modulating the sensitivity and dynamics of Ire1 activity.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Transient Ire1 activation in non-lethal ER stress conditions.
(A) After depletion of inositol from the growth media, wild type yeast cells were sampled from a master culture every 20 min, and total RNA was purified and subjected to Northern blot analysis using a probe for the first exon of HAC1 mRNA. After a lag phase, HAC1 mRNA splicing displayed activation and deactivation phases. u, unspliced HAC1 mRNA; s, spliced HAC1 mRNA. Right panel: wild type cells 0 min and 240 min after inositol depletion and probed for the INO1 mRNA. (B) Cell growth was monitored over time in wild type cells treated with 5 mM, 2.2 mM, 1.5 mM, and 0 mM DTT by measuring the OD600. Cells treated with 5 mM DTT cease to divide, while cells treated with 2.2 mM or 1.5 mM DTT continue to grow. (C) Wild type cells were treated with 5 mM, 2.2 mM, or 1.5 mM DTT and sampled over time. After Northern blot analysis, the percentage of spliced HAC1 mRNA was quantified (blots are shown in the supplement). Cells treated with 5 mM DTT displayed sustained maximal splicing, while cells treated with 2.2 mM or 1.5 mM displayed transient HAC1 mRNA splicing: the same activation and deactivation phases as the response to the depletion of inositol. (D) Wild type cells were constructed bearing a transcriptional reporter (TR) consisting of four repeats of a UPR-responsive DNA element controlling the expression of GFP. These cells were treated with 2.2 mM, 1.5 mM, or 0 mM DTT, sampled over time, and subjected to flow cytometry to quantify the GFP fluorescence. The TR was induced to dose-dependent plateaus due to the >8 h half life of GFP. % max is defined as the GFP fluorescence in cells treated with 5 mM DTT for 4 h. (E) When plotted as the rate of GFP produced per minute, the TR displayed the same activation and deactivation phases as spliced HAC1 mRNA. Transient Ire1 activation leads to transient transcriptional activation. % max as defined in (D).
Figure 2
Figure 2. Ire1bipless is stress-activated with no change to its association with BiP.
(A) Ire1bipless is a mutant of Ire1 lacking 51 amino acids containing the BiP interaction motif (Δ475–526). Cells bearing HA-tagged alleles of wild type Ire1 or Ire1bipless were harvested before and after treatment with 5 mM DTT for 1 h. Cells were lysed and Ire1 and Ire1bipless were immuno-precipitated with anti-HA agarose beads. The proteins eluted from the beads were resolved by SDS-PAGE, transferred to PVDF, co-incubated with anti-HA and anti-BiP antibodies followed by fluorophore-conjugated secondary antibodies, and scanned on the Li-Cor imager. BiP decreased its association with wild type Ire1 after treatment with DTT, while BiP did not change its association with Ire1bipless after DTT treatment. Some BiP binds nonspecifically. (B) Three independent immunoprecipitation experiments were quantified after scanning with the Li-Cor. The ratio of BiP/Ire1, after subtraction of the nonspecific BiP signal as measured in the Ire1Δ cells, shows that BiP dissociates from wild type Ire1 in response to DTT, that Ire1bipless binds to less BiP in the absence of stress than wild type Ire1 binds in the presence of DTT, and that Ire1bipless does not change its association with BiP after treatment with DTT. (C) Cells bearing wild type Ire1 or Ire1bipless were harvested before and after treatment with 5 mM DTT for 1 h, total RNA was purified, subjected to Northern blot analysis, and probed for HAC1 mRNA. Wild type and Ire1bipless displayed no differences in splicing: no HAC1 mRNA was spliced in the absence of DTT and splicing was equally induced after treatment with DTT. (D) GFP-tagged alleles of wild type Ire1 and Ire1bipless were expressed and imaged in the presence and absence of DTT. GFP domains are inserted between the transmembrane domain and the linker of the kinase domain on the cytoplasmic side of Ire1, as in . Wild type Ire1 displays a diffuse perinuclear and cortical ER localization in the absence of stress and forms bright clusters after treatment of 5 mM DTT for 1 h. Ire1bipless displays similar perinuclear and cortical localization in the absence of stress, but with small clusters in some cells. After DTT treatment, Ire1bipless forms clusters like the wild type. (E) Quantification of Ire1 clustering shows that Ire1bipless forms more foci in the absence of stress than wild type, but forms clusters equal to the wild type after treatment with 5 mM DTT for 1 h. (F) Wild type and irebipless cells in the absence of stress probed for basal expression of INO1 mRNA expression.
Figure 3
Figure 3. Experimental and simulated DTT titration time courses in wild type, hac1Δ, and Ire1bipless cells.
(A) Wild type cells expressing the GFP splicing reporter (SR) were treated with doses of DTT spanning the active concentration range, sampled over time, and their fluorescence was measured by flow cytometry. The SR, like the TR, reached dose-dependent plateaus due to the >8 h half life of GFP. (B) hac1Δ cells expressing the SR were treated as above. hac1Δ cells were hypersensitive to DTT and saturate the reporter at all experimental doses. (C) Ire1bipless cells expressing the SR were treated as above and showed increased sensitivity to DTT compared to the wild type, responding to 0.66 mM DTT and saturating at 1.5 mM DTT. (D) Simulations of the “wild type” model. The architecture of the model, described in the text and depicted in Figure 4A, includes BiP binding to Ire1 and negative feedback. When the model includes a cooperative Ire1 deactivation term (described in text), it recapitulated the wild type DTT titration time course. (E) Simulations of the “hac1Δ” in which the negative feedback terms have been removed captured the hypersensitivity observed experimentally. (F) Simulations of the “Ire1bipless” model in which the Ire1/BiP interaction terms have been removed revealed the increased DTT sensitivity compared to the wild type.
Figure 4
Figure 4. Model architecture, prediction and experimental validation.
(A) The molecular interactions that comprise the model. See the supplement for complete modeling details. Ire1 can exist in three states: (1) inactive monomer (Ire1i, middle lower box), (2) inactive in complex with BiP (Ire1i•BiP, middle lower box), and (3) active in complex with an unfolded protein (Ire1a•UP, lower right box). Either reduced (UPr) or oxidized (UPo) can bind to and activate Ire1, but UPos quickly become folded proteins (FP, upper box and lower left box). The amount of UPrs and UPos is determined by the flux of unfolded proteins and the red/ox potential, defined here as the ratio of Ero1/DTT. Active Ire1 in complex with unfolded proteins produces the Hac1 transcription factor, which induces the production of Ero1 and BiP. BiP can also exist in three states: (1) monomer (BiP, middle lower box), (2) bound to Ire1i (BiP•Ire1i), and (3) in complex with unfolded proteins (BiP•UP). BiP can bind to both UPr and UPo, but only aids in the folding of UPo (bottom left box). The blue arrows indicate the feedback terms that are removed in the “hac1Δ” model, and the red arrows indicate the Ire1/BiP interaction terms that are removed in the “Ire1bipless” model. (B) Simulations “wild type” and “Ire1bipless” cells treated with 5 mM DTT for 100 min and then the DTT is suddenly removed predict a deactivation delay for Ire1bipless cells: “wild type” cells immediately began to deactivate while Ire1bipless continued activity for ∼30 min after DTT withdrawal. (C) Wild type and Ire1bipless were treated with 5 mM DTT for 1 h, filtered, washed, and resuspended in fresh media lacking DTT and sampled over time. Samples were assayed for HAC1 mRNA splicing by Northern blot to measure Ire1 activity. Consistent with the simulations, wild type cells deactivated after 90 min while Ire1bipless cells deactivated after 180 min.
Figure 5
Figure 5. FRET measurements of wild type Ire1 and Ire1bipless.
(A) Cartoon of Ire1 FRET. GFP- and mCherry-tagged versions of Ire1 or Ire1bipless were co-expressed and cells were imaged by confocal microscopy. GFP and mCherry domains are inserted between the transmembrane domain and the kinase linker on the cytoplasmic side of Ire1, as in . When exposed to blue light (488 nm) the GFP is excited, and if it is within a few nm of mCherry, it can excite mCherry instead of emitting green light. This transferred energy is emitted by mCherry as red light and can be measured as a FRET signal. (B) DTT titration time course measured by FRET in wild type cells. Ire1 displayed transient oligomerization after treatment with 2.2 mM or 1.5 mM DTT, and sustained oligomerization in response to 5 mM DTT. Doses are indicated in (C). (C) DTT titration time course measured by FRET in Ire1bipless cells. Ire1bipless displayed sustained oligomerization after treatment with 2.2 mM or 1.5 mM DTT, and transient activation after treatment with 0.66 and 0.99 mM DTT. (D) Cells expressing FRET pairs of wild type Ire1 (top panels) or Ire1bipless (bottom panels) were treated with 5 mM DTT for 1 h and subsequently washed, resuspended in fresh media, and imaged by confocal microscopy. (E) Quantification of FRET signal from DTT washout experiment. Wild type Ire1 de-oligomerized completely by 90 min, while Ire1bipless did not fully de-oligomerize for 180 min.
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