(A) Steady state protein levels and localization of Opi1, Ino2, and Ino4 under repressing and activating conditions. Strains expressing myc-tagged Opi1, Ino2, or Ino4 (Longtine et al. 1998) were grown in the presence (INO1 repressing condition) or absence (INO1 activating condition) of myo-inositol for 4.5 h. Tagged proteins were analyzed by Western blotting (size-fractionated blots on the left, designated Opi1, Ino2, and Ino4) and indirect immunofluorescence (photomicrographs on the right). For Western blot analysis, 25 μg of crude lysates were immunoblotted using monoclonal antibodies against either the myc epitope (top bands in each set) or, as a loading control, Pgk1 (bottom bands in each set; indicated with an asterisk). Immunofluorescence experiments were carried out using anti-myc antibodies and anti-mouse Alexafluor 488. Bright-field (BF) and indirect fluorescent (IF) images for a single z slice through the center of the cell were collected by confocal microscopy.
(B) Ino2 and Ino4 heterodimerize under both repressing and activating conditions. Cells expressing either HA-tagged Ino4 (negative control) or HA-tagged Ino4 and myc-tagged Ino2 were grown in the presence or absence of 1 μg/ml tunicamycin (Tm; an inhibitor of protein glycosylation that induces protein misfolding in the ER) for 4.5 h and lysed. Proteins were immunoprecipitated using the anti-myc monoclonal antibody. Immunoprecipitates were size-fractionated by SDS-PAGE and immunoblotted using the anti-HA monoclonal antibody. (Continued on next page)
(C) Coimmunoprecipitation of Scs2 with Opi1. Detergent-solubilized microsomal membranes from either an untagged control strain (lane C) or duplicate preparations from the Opi1-myc tagged strain (myc lanes, 1 and 2) were subjected to immunoprecipitation using monoclonal anti-myc agarose. Immunoprecipitated proteins were size-fractionated by SDS-PAGE and stained with colloidal blue. Opi1-myc and the band that was excised and identified by mass spectrometry as Scs2 are indicated. IgG heavy and light chain bands are indicated with an asterisk.
(D) Coimmunoprecipitation with tagged proteins. Immunoprecipitation analysis was carried out on strains expressing either Scs2-HA alone (lanes 1–3) or Scs2-HA together with Opi1p-myc (lanes 4–6). Equal fractions of the total (T), supernatant (S), and bound (B) fractions were size-fractionated by SDS-PAGE and immunoblotted using anti-myc or anti-HA monoclonal antibodies.
(E) Epistasis analysis. Haploid progeny from an OPI1/opi1ΔSCS2/scs2Δ double heterozygous diploid strain having the indicated genotypes were streaked onto minimal medium with (+ inositol) or without (– inositol) 100 μg/ml myo-inositol and incubated for 2 d at 37 °C.

(A) Untagged control cells (upper images), or cells in which the endogenous copies of INO2 and INO4 were replaced with HA-tagged Ino2 (center images) or HA-tagged Ino4 (lower images) were harvested in mid-logarithmic phase and washed into medium with or without myo-inositol. After 4.5 h, about 1.5 × 10⁸ cells were harvested and processed for Northern blot analysis (light images with dark bands, right). Northern blots were probed against both INO1 and ACT1 (loading control) mRNA. The remaining cells were fixed with formaldehyde and lysed. Chromatin was sheared by sonication and then subjected to immunoprecipitation with anti-HA agarose. Input DNA (In) and immunoprecipitated DNA (IP) were analyzed by PCR using primers to amplify the INO1 promoter and the URA3 gene. Amplified DNA was size-fractionated by electrophoresis on ethidium bromide-stained agarose gels (dark images with light bands, left).
(B) Quantitative PCR analysis. Input and IP fractions were analyzed by real-time quantitative PCR. The ratio of INO1 promoter to URA3 template in the reaction is shown. Error bars represent the standard error of the mean (SEM) between experiments.

(A) Chromatin-associated Opi1 dissociates upon activation of the UPR. Cells of the indicated genotypes were harvested after growth for 4.5 h with or without myo-inositol, fixed, and processed as in Figure 2. The scs2Δ mutant was transformed with pRS315-Opi1-myc, a CEN ARS plasmid that expresses Opi1-myc at endogenous levels. Input DNA (In) and immunoprecipitated DNA (IP) were analyzed by PCR using primers to amplify the INO1 promoter and the URA3 gene. Amplified DNA was separated by electrophoresis on ethidium bromide–stained agarose gels.
(B) Quantitative PCR analysis. Input and IP fractions were analyzed by real-time quantitative PCR. The ratio of INO1 promoter to URA3 template in the reaction is shown. Error bars represent the SEM between experiments.

The carboxyl-terminal transmembrane domain of Scs2 was removed by replacement with three copies of the HA epitope (Scs2ΔTMD-HA; Longtine et al. [1998]).
(A) Scs2ΔTMD localization. Ribosomal protein S2 (Rps2-HA), Scs2-HA, and Scs2ΔTMD-HA were localized by immunofluorescence against the HA epitope. DNA was stained with 4′,6′-diamidino-2-phenylindole. Images were collected in a single z-plane (≤ 0.7 μm thick) by confocal microscopy. Unlike Rps2-HA, which was excluded from the nucleus (indicated with white arrows), Scs2ΔTMD-HA staining was uniform and evident in the nucleoplasm.
(B) Scs2ΔTMD steady-state levels. Equal amounts of whole cell extract from cells expressing either Scs2-HA or Scs2ΔTMD-HA were analyzed by immunoblotting.
(C) Scs2ΔTMD is nonfunctional. Strains expressing the indicated forms of Scs2 were streaked onto medium with or without myo-inositol and incubated for 2 d at 37 °C.

An array of Lac operator repeats was integrated at INO1 or URA3 in strains expressing GFP-Lac repressor and myc-tagged Sec63. GFP-Lac repressor and Sec63-myc were localized in fixed cells by indirect immunofluorescence. Data were collected from single z sections representing the maximal, most focused signal from the Lac repressor.
(A) Two classes of subnuclear localization. Shown are five representative examples of localization patterns that were scored as membrane-associated (photomicrographs and plots on left) or nucleoplasmic (right). For each image, the fluorescence intensity was plotted for each channel along a line that intersects both the Lac repressor spot and the center of the nucleus.
(B) INO1 is recruited to the nuclear membrane upon activation. The fraction of cells that scored as membrane-associated is plotted for each strain grown in the presence (+) or absence (–) of inositol. The site of integration of the Lac operator (Lac O), the version of the GFP-Lac repressor (GFP-Lac I; either wild-type or having the FFAT membrane-targeting signal) expressed, and the relevant genotype of each strain is indicated. The dashed line represents the mean membrane association of the URA3 gene. The vertical arrow indicates the frequency of membrane association in the wild-type strain under activating conditions. Error bars represent the SEM between separate experiments. Each experiment scored at least 30 cells. The total number of cells (and experiments) scored for each column were: bar 1, 70 (2); bar 2, 66 (2); bar 3, 39 (1); bar 4, 71 (2); bar 5, 140 (4); bar 6, 88 (2); bar 7, 88 (2); bar 8, 92 (3); bar 9, 74 (2); and bar 10, 38 (1).

(A) Northern blot analysis of membrane-targeted INO1. Strains of the indicated genotypes having the Lac operator array integrated at INO1 and expressing either the wild-type GFP-Lac repressor or GFP-FFAT-Lac repressor were grown in the presence or absence of 1 μg/ml tunicamycin (Tm) for 4.5 h, harvested, and analyzed by Northern blot. Blots were probed for either INO1 or ACT1 (as a loading control) mRNA. The wild-type strain CRY1, lacking both the Lac operator array and the Lac repressor, was included in the first two lanes for comparison.
(B) Wild-type or scs2Δ mutant strains in which the Lac operator had been integrated at INO1 were transformed with either GFP-Lac repressor or GFP-FFAT-Lac repressor. The resulting transformants were serially diluted (tenfold between wells) and spotted onto medium lacking inositol, uracil, and histidine, and incubated for 2 d at 37 °C.
(C) Wild-type and scs2Δ mutant strains transformed with either GFP-Lac repressor or GFP-FFAT-Lac repressor, but lacking the Lac operator, were streaked onto medium lacking inositol and histidine and incubated for 2 d at 37 °C.

Ino2 and Ino4 bind constitutively to the INO1 promoter. Under repressing conditions, Opi1 associates with chromatin to prevent activation, and the INO1 locus localizes to the nucleoplasm. Hac1 synthesis under UPR-inducing conditions promotes dissociation of Opi1 from chromatin. Scs2 binds to Opi1 at the nuclear membrane to stabilize the non-chromatin-bound state. Dissociation is coupled to recruitment of INO1 to the nuclear membrane, where transcriptional activation occurs.