BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response
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 [13]. 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.