1. Volume 6, Issue 6, Pages 1355-1364 (December 2000)
ER Stress Induces Cleavage of Membrane-Bound ATF6 by the Same Proteases that Process SREBPs 
Jin Ye, Robert B Rawson, Ryutaro Komuro, Xi Chen, Utpal P Davé, Ron Prywes, Michael S Brown, Joseph L Goldstein 
Molecular Cell 
Volume 6, Issue 6, Pages (December 2000)
DOI: /S (00)

2. Figure 1
Comparison of Amino Acid Sequences of Human SREBP-2 and Human ATF6 in Region of Regulated Intramembrane Cleavage
The sequences are aligned without the introduction of gaps. The box denotes the position of the first transmembrane domain of SREBP-2. Amino acids that are necessary for Site-1 and Site-2 cleavage of SREBP-2 are highlighted in orange and green, respectively. Amino acids that potentially play the same role in the proteolytic cleavage of ATF6 are numbered and highlighted in the same way.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Proteolytic Processing and Transcriptional Activity of ATF6 Are Stimulated by Tunicamycin but Not by Sterol Depletion in Transfected HEK-23 Cells
In (A), HEK-293 cells were set up for experiments on day 0 and transfected on day 2 as described in Experimental Procedures with 2 μg/dish of pTK-HSV-ATF6 or empty-vector pcDNA 3.1 (Invitrogen) as indicated. Three hours after transfection, cells were switched to medium containing 5% fetal calf serum. On day 3, cells received a direct addition of 25 μg/ml ALLN in the absence or presence of 2 μg/ml tunicamycin as indicated. Four hours later, cells were harvested and fractionated as described in Experimental Procedures. In (B), HEK-293 cells were transfected on day 2 with 2 μg/dish of pTK-HSV-ATF6 or empty vector pCDNA3.1 as indicated. After transfection, cells were switched to medium containing either 5% fetal calf serum (FCS) or 5% newborn calf lipoprotein-deficient serum containing 50 μM compactin and 50 μM sodium mevalonate (LPDS) in the absence or presence of 1 μg/ml of 25-hydroxycholesterol plus 10 μg/ml cholesterol (sterols) as indicated. On day 3, cells received a direct addition of 25 μg/ml ALLN in the absence or presence of 2 μg/ml of tunicamycin as indicated. After incubation for 4 hr, the cells were harvested and fractionated. In (A )and (B), aliquots of nuclear extracts (30 μg protein) and membranes (10 μg protein) were subjected to SDS–PAGE and immunoblot analysis as described in Experimental Procedures. Filters were exposed for 7 s. P, P*, I, and N denote the precursor, precursor with no glycosylation, intermediate, and nuclear forms of HSV-tagged ATF6, respectively. In (C) and (D), HEK-293 cells were set up on day 0 at a density of 1 × 105 cells per 22 mm well. On day 2, the cells were transfected with 1 μg/dish of either p5xATF6GL3 (C) or pSRE-luciferase (D) together with 0.05 μg/dish of pCMV β-Gal using an MBS kit as described in Experimental Procedures. Three hours after transfection, cells were switched to medium containing 5% newborn calf lipoprotein-deficient serum containing 50 μM compactin and 50 μM sodium mevalonate in the absence or presence of 2 μg/ml of tunicamycin and/or 1 μg/ml of 25-hydroxycholesterol plus 10 μg/ml cholesterol (sterols) as indicated. After incubation for 16 hr, cells were harvested, and luciferase activity was measured and normalized to β-galactosidase activity as described in Experimental Procedures. A value of 1 represents the normalized luciferase activity in cells not treated with either tunicamycin or sterols. Each value represents the average (and range) of duplicate incubations. Similar results were obtained in one other independent experiment.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
Proteolytic Processing and Transcriptional Activity of ATF6 in Wild-Type and Mutant CHO Cells Lacking S1P, S2P, or SCAP
(A) Proteolytic processing. The indicated cells were set up and transfected with 2 μg/dish of pTK-HSV-ATF6 or empty-vector pCDNA3.1 as described in Experimental Procedures. After 20 hr, cells were treated with or without 2 μg/ml of tunicamycin as indicated. Four hours later, the cells were harvested and fractionated, and aliquots of nuclear extracts (30 μg protein) and membranes (10 μg protein) were subjected to SDS–PAGE and immunoblot analysis. Filters were exposed for 15 s. P, P*, X, I, and N denote the precursor, precursor with no glycosylation, band X, intermediate, and nuclear forms of HSV-tagged ATF6, respectively.
(B) Transcriptional activity. On day 0, cells were set up at a density of 1 × 105 cells per 22 mm well. On day 1, the cells were transfected with p5xATF6GL3 (1 μg/dish) and pCMV β-Gal (0.05 μg/dish) using the Fugene-6 reagent as described in Experimental Procedures. After 10 hr, the cells were treated with or without 2 μg/ml of tunicamycin as indicated. Sixteen hours later, the cells were harvested, and luciferase activity was measured and normalized to β-galactosidase activity as described in Experimental Procedures. A value of 1 represents the normalized luciferase activity in cells not treated with tunicamycin. Each value represents the average (and range) of duplicate incubations. Similar results were obtained in two other independent experiments.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Transfected S1P Stimulates Proteolytic Processing of ATF6 in Mutant CHO Cells Lacking S1P
S1P-deficient SRD-12B cells were cotransfected with a plasmid encoding HSV-tagged ATF6 (2 μg/dish) together with 0.5 μg/dish of either wild-type S1P (WT) or mutant S1P (S414A) as indicated. The total amount of DNA was adjusted to 2.5 μg/dish by addition of pCDNA3.1 empty vector. After 20 hr, cells were treated with or without 2 μg/ml tunicamycin as indicated. Four hours later, the cells were harvested and fractionated, and aliquots of nuclear extracts (30 μg protein) and membranes (10 μg protein) were subjected to SDS–PAGE and immunoblot analysis as described in Experimental Procedures. Filters were exposed for 15 s. P, P*, X, I, and N denote the precursor, precursor with no glycosylation, band X, intermediate, and nuclear forms of HSV-tagged ATF6, respectively.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5
Mutation Altering S1P Recognition Site in ATF6 Reduces Its Proteolytic Processing in Transfected HEK-293 Cells
HEK-293 cells were set up and transfected with a plasmid encoding either wild-type or mutant HSV-tagged ATF6 (2 μg/dish) as indicated. After 20 hr, cells were treated with or without 2 μg/ml of tunicamycin as indicated. Four hours later, the cells were harvested and fractionated, and aliquots of nuclear extracts (30 μg protein) and membranes (10 μg protein) were subjected to SDS–PAGE and immunoblot analysis. Filters were exposed for 7 s. P, P*, X, I, and N denote the precursor, precursor with no glycosylation, band X, intermediate, and nuclear forms of HSV-tagged ATF6, respectively.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6
Transfected S2P Restores Proteolytic Processing and Transcriptional Activity of ATF6 in Mutant CHO Cells Lacking S2P
(A) Proteolytic processing. M19 cells were cotransfected with a plasmid encoding HSV-tagged ATF6 (2 μg/dish) together with 0.5 μg/dish of either wild-type (WT) or mutant S2P (H171F) as indicated. The total amount of DNA was adjusted to 2.5 μg/dish by addition of pCDNA3.1 empty vector. After 20 hr, cells were treated with or without 2 μg/ml of tunicamycin as indicated. Four hours later, the cells were harvested and fractionated, and aliquots of nuclear extracts (30 μg protein) and membranes (10 μg protein) were subjected to SDS–PAGE and immunoblot analysis. Filters were exposed to film for 15 s. P, P*, I, and N denote the precursor, precursor with no glycosylation, intermediate, and nuclear forms of HSV-tagged ATF6, respectively.
(B) Transcriptional activity. Cells were set up, transfected, incubated, and harvested for luciferase assay as described in the legend to Figure 3B. Each value represents the average (and range) of duplicate incubations. Similar results were obtained in two other independent experiments.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
Asparagine and Proline within Membrane-Spanning Segment of HSV-ATF6 Are Required for Its Proteolytic Processing in Transfected HEK-293 Cells
HEK-293 cells were set up and transfected with a plasmid encoding either wild-type or mutant HSV-tagged ATF6 (2 μg/dish) as indicated. Cells were then incubated and harvested for immunoblot analysis as described in the legend to Figure 5. Filters were exposed to film for 15 s. P, P*, I, and N denote the precursor, precursor with no glycosylation, intermediate, and nuclear forms of HSV-tagged ATF6, respectively.
Molecular Cell 2000 6, DOI: ( /S (00) )

9. Figure 8
Treatment with Tunicamycin or Thapsigargin Fails to Induce GRP78 (BIP) in Mutant CHO Cells Deficient in S2P
(A) Immunoblot analysis of GRP78. On day 0, CHO-7 cells (lanes 1–3), M19 cells (lanes 4–6), and M19 cells stably transfected with either HSV-tagged wild-type S2P (lanes 7–9) or HSV-tagged mutant S2P (H171F) (lanes 10–12) were set up at a density of 1 × 106 per 60 mm dish. On day 1, the cells received either tunicamycin (2 μg/ml) or thapsigargin (300 nM) as indicated. After incubation for 16 hr, the cells were harvested and fractionated, and aliquots of membranes (20 μg protein) were subjected to SDS–PAGE and immunoblot analysis with anti-KDEL antibody as described in Experimental Procedures. Filters were exposed to film for 2 s.
(B) Immunoblot analysis of S2P. Aliquots of membranes (20 μg protein) from lanes 4, 7, and 10 in (A) were subjected to SDS–PAGE and immunoblot analysis with anti-HSV-Tag antibody to visualize wild-type and mutant S2P. Filters were exposed to film for 15 s.
Molecular Cell 2000 6, DOI: ( /S (00) )