Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2+
- Degenerative Diseases Program, SBP Medical Discovery Institute, United States
- Gottfried Schatz Research Center, Medical University of Graz, Austria
- University of California, San Diego, United States
Figures
Figure 1 with 8 supplements
ER ATP homeostasis is maintained by oxidative phosphorylation.
(A) Confocal microscopy confirms ER localization of the ER ATP FRET reporter, ERAT in H9-D2 CHO cells. A representative confocal micrograph shows a high degree of co-localization of ERAT …
Figure 1—figure supplement 1
The ERAT probe is localized to the ER lumen.
(A) Confocal microscopy confirms ER localization of the ER ATP reporter, ERAT, in fixed H9 CHO cells. A representative confocal micrograph shows a high degree of co-localization of ERAT fluorescence …
Figure 1—figure supplement 2
The ERAT probe is localized to the ER lumen.
(A) Confocal microscopy confirms ER localization of the ER ATP reporter, ERAT, in live H9-D2 CHO cells. A representative confocal micrograph shows a high degree of co-localization of ERAT …
Figure 1—figure supplement 3
A single clone of H9-D2 cells was engineered to facilitate flow-cytometry based ER ATP analysis.
After ERAT4N7Q transfection and G418 selection, a single clone of H9 cells, H9-D2 cells, was further obtained to facilitate the flow-based FRET assay. H9-D2 cells have unanimous ERAT4N7Q expression …
Figure 1—figure supplement 4
2-DG treatment does not reduce OxPhos of H9 CHO cells.
(A) 2-DG treatment for ~ 30 min (20 mM final concentration) has no effect on cellular oxygen usage. Oxygen consumption rates (OCR), in H9 CHO cells were measured by an XF-24 platform before serial …
Figure 1—figure supplement 5
ER localization of ERAT probe is not affected by brief treatment with bio-energetic inhibitors.
Confocal microscopy confirms ER localization of the ERAT reporter in live H9-D2 CHO cells, both before and after treatment with bio-energetic inhibitors for 30 min. A representative confocal …
Figure 1—figure supplement 6
The dynamic range of the ER ATP change was determined by flow cytometry.
In an effort to determine the dynamic range of the FRET ratio in response to ER ATP change, H9 CHO cells were first treated with DMSO (A). (1% by volume) or 2-DG (B). (20 mM) for 2 hr, before the …
Figure 1—figure supplement 7
Glucose supplementation does not alter ER ATP levels and 3-Bromopyruvate increases ER ATP levels.
(A) Glucose supplementation (5 mM x 1 hr) in basal medium with only OxPhos substrates has no effect on ATP levels in the ER, while oligomycin reduces ER ATP. The glucose supplemented group is …
Figure 1—figure supplement 8
Glucose supplementation increases total cellular ATP while 2-DG decreases the cytosolic ATP-to-ADP ratio.
(A) Glucose supplementation (5 mM x1 hr) in basal medium with only OxPhos substrates increases the total cellular ATP content. Basal medium is DMEM medium with only OxPhos substrates (Seahorse …
Figure 2 with 2 supplements
A Ca2+ gradient across the ER membrane is required to maintain ER ATP homeostasis.
(A) SERCA inhibition reduces ER ATP levels in adherent INS1 cells. BHQ (15 µM) was added at 0 min as indicated. Scale bar represents 10 µm. (B) A representative trace of the FRET signal overlaid …
Figure 2—figure supplement 1
ER ATP levels in INS1 cells decrease after BHQ-mediated SERCA inhibition and recovery takes time.
(A) Experimental scheme is shown for panels B) and C). Briefly, INS-1 cells were first treated with 15 µM BHQ for 30 min to reduce ER ATP levels, and after BHQ removal, the cells were allowed to …
Figure 2—figure supplement 2
Tg and CPA decrease ER Ca2+ and ATP in H9 CHO cells due to SERCA inhibition.
(A) Treatment with cyclopiazonic acid (CPA, at 50 µM) or Tg (1 µM) for 18 min decreases ER ATP levels. ER ATP was estimated by flow cytometry using the ERAT reporter. (B) ER Ca2+ levels in H9 CHO …
Figure 3 with 6 supplements
ATP transport from mitochondria to ER is inhibited by cytosolic Ca2+.
ATP stores in plasma membrane permeabilized H9 CHO cells were measured by a reporter-free method. H9 CHO cells were permeabilized with 75 µg/mL saponin in a bathing solution (referred to as …
Figure 3—figure supplement 1
Permeabilized H9 CHO cells produce ATP through OxPhos and ATP is detected in the respiration buffer.
(A) Ca2+ supplementation does not alter mitochondrial respiration of PM-permeabilized H9 CHO cells. Oxygen consumption rate (OCR in pMole/min) was measured using a Seahorse XF-24 platform. For the …
Figure 3—figure supplement 2
In complement to Figure 3E.
(A) ATP produced and partitioned to the supernatant was measured using ATPLite assay. For the scrambled siRNA control group, ATP content in the supernatant was significantly higher in the ‘1 µM Ca2+’…
Figure 3—figure supplement 3
Similar to the result in Figure 3F,.
(A) The Ca2+ responsiveness of ER ATP levels by SERCA inhibition was attenuated by siRNA-mediated Slc35b1 knock-down in INS1 cells. When the baseline and endpoint FRET ratios were quantified, …
Figure 3—figure supplement 4
Slc35b1 transcript was quantified by qRT-PCR, and the knock-down efficiency was > 90% in HeLa cells (A), and ~ 33% in INS1 cells (B).
https://doi.org/10.7554/eLife.49682.018
Figure 3—figure supplement 5
The CaATiER mechanism responds to cytosolic Ca2+ in a physiological range.
(A) ER ATP is reduced in response to cytosolic Ca2+ in the low µM range. (B) ER ATP levels were expressed as percentage of total ATP. (C) In contrast to ER ATP shown in (A), ATP contained in the …
Figure 3—figure supplement 6
Since the respiration buffer contains 5 mM MgCl2 as a basal ingredient, an additional 5 to 10 mM MgCl2 was added to final concentrations of ‘10 mM’ and ‘15 mM’ as indicated, to test the Mg2+ effect on ER ATP.
The difference between ‘5 mM’ and ‘10 mM’ was not statistically significant (p=0.21), while EGTA supplemented at 1 mM had a significant effect (p<0.0001) compared to 5 mM Mg2+ with ‘no EGTA’.
Figure 4 with 2 supplements
Protein misfolding in the ER increases ER ATP dependence on OxPhos and increases ER to mitochondrial Ca2+ trafficking.
(A) When treated with HDAC inhibitors, such as SAHA or sodium butyrate (NaB), F8 transcription is induced in H9 CHO cells accompanied by UPR activation. SAHA was tested at increasing concentrations, …
Figure 4—figure supplement 1
While mitochondrial matrix ATP levels remain unchanged in ER stressed H9 cells, upon OxPhos inhibition the cytosolic ATP/ADP ratios decrease more quickly than unstressed H9 cells.
Mitochondrial ATP levels are not changed in un-induced H9 CHO cells (A) and SAHA-treated H9 cells to induce F8 expression (B). Cells were equally sensitive to 1 µM oligomycin. ATP levels were …
Figure 4—figure supplement 2
Glucose uptake in ER stressed H9 CHO cells was reduced compared to non-stressed H9 CHO cells.
The uptake of a fluorescent glucose analog, 2-NBDG, was quantified by flow-cytometry and compared between non-stressed versus ER stressed H9 CHO cells. (A) With overnight NaB incubation, ER stressed …
Author response image 1
Author response image 2
Left panel – Illustration of linkers used; Right panel – OCR results before and after linkers ligation by Rapa.
DT-8 = Double transfected clone 8, with both linkers present, experimental group. AR-6/-10* = AKAP1-mRFP, with only mito-linker present, clone-6/-10. *AR clones served as control clones.
Author response image 3
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Cricetulus griseus) | CHO DUK cell line | Dr. Lawrence Chasin at Columbia University, USA., Personal gift | N/A | Confirmed by karyotyping |
| Cell line (Cricetulus griseus) | CHO H9 cell line | Made in Kaufman Lab | N/A | Confirmed by karyotyping, further by F8 WB |
| Cell line (Rattus norvegicus) | INS1 cell line | Gift from Dr. Christopher B. Newgard, Duke University, USA | N/A | Confirmed by WB and electrophysiology |
| Cell line (Homo-sapiens) | HeLa S3 cell line | ATCC | # CCl-2.2 | Assurance by ATCC |
| Antibody | anti-F8, IgG1 (Mouse monoclonal) | Green Mountain Antibodies | GMA 012 | WB: 1:1000 |
| Antibody | anti-BiP, IgG (Rabbit monoclonal) | Cell Signaling Technology | CST 3177 | WB: 1:1000 |
| Antibody | anti-CHOP, IgG (Rabbit polyclonal) | Santa Cruz Biotechnology | SC 575 | WB: 1:1000 |
| Antibody | phospho-eIF2α (Ser51), IgG (Rabbit monoclonal) | Cell Signaling Technology | CST 3597S | WB: 1:1000 |
| Antibody | anti-phospho-p70S6K (Thr421/Ser424), IgG (Rabbit polyclonal) | Cell Signaling Technology | CST 9204 | WB: 1:1000 |
| Antibody | anti-VINCULIN, IgG1 (Mouse monoclonal) | Sigma | V9131 | WB: 1:1000 |
| Recombinant DNA reagent | mtAT 1.03, plasmid | Dr. Hiromi Imamura at Kyoto University, personal gift | N/A | ATP level |
| Recombinant DNA reagent | ERAT 4.01 N7Q, plasmid | NGFI, Austria | N/A | ATP level |
| Recombinant DNA reagent | pTagRFP-C, plasmid | Evrogen | #FP141 | Cytosolic location |
| Recombinant DNA reagent | GW1-Perceval HR, plasmid | Addgene | #49082 | ATP/ADP ratio |
| Recombinant DNA reagent | D1ER, plasmid | Dr. Demaurex at Universitè de Genève, Switzerland, personal gift | N/A | Ca2+ level |
| Recombinant DNA reagent | pCIS GEM-CEPIA1er, plasmid | Addgene | #58217 | Ca2+ level |
| Recombinant DNA reagent | CMV-mito-mtGEM-GECO1, plasmid | Addgene | #32461 | Ca2+ level |
| Recombinant DNA reagent | ER-RFP, plasmid | Addgene | #62236 | ER localization |
| Recombinant DNA reagent | cpYFP, plasmid | Dr. Yi Yang at East China University of Science and Technology, personal gift | N/A | pH level |
| Sequence-based reagent | Puromycin resistant gene, 5’ primer 5’-ACAAATGTGGTAAAATCGATAAGGATCCG-3’; | Integrated DNA Technologies | N/A | PCR primer |
| Sequence-based reagent | Puromycin resistant gene, 3’ primer 5’-GAGCTGACTGGGTTGAAGGCT-CTCAAGGGC-3’ | Integrated DNA Technologies | N/A | PCR primer |
| Sequence-based reagent | siRNA Slc35b1- 5’-GAG ACU ACC UCC ACA UCA A dTdT-3’ (targeting 3’-UTR of human gene) | Microsynth AG, Balgach, Switzerland | N/A | siRNA |
| Sequence-based reagent | siRNA scrambled- 5'-AGG UAG UGU AAU CGC CUU G dTdT-3' (control for humanSlc35b1 knockdown) | Microsynth AG, Balgach, Switzerland | N/A | siRNA |
| Sequence-based reagent | siRNA targeting mouse Slc35b1 sequence – 5’-CCACATGATGTTGAACATCAA-3’ | Qiagen | Mm_Ugalt2_1, SI01461523 | siRNA |
| Sequence-based reagent | siRNA targeting mouse Slc35b1 sequence – 5’- AAGAAGGTGGTTGGAATAGAA-3’ | Qiagen | Mm_Ugalt2_2, SI01461530 | siRNA |
| Sequence-based reagent | siRNA targeting mouse Slc35b1 sequence – 5’-TCGGTAAATCCTGCAAGCCAA-3’ | Qiagen | Mm_Ugalt2_4, SI01461544 | siRNA |
| Sequence-based reagent | siRNA scrambled – Sequence proprietary | Qiagen | Allstars Negative Control siRNA, Cat #1027280 | siRNA |
| Commercial assay or kit | TransFast Transfection Reagent | Promega Corporation, Madison, USA | # E2431 | |
| Commercial assay or kit | FuGENE 6 Transfection Reagent | Promega | # E2693 | |
| Commercial assay or kit | Calcium Assay Kit | Adipogen Corp. | # JAI-CCA-030 | |
| Commercial assay or kit | XF24 extracellular flux assay kit | Seahorse BioSciences | # 100850–001 | |
| Commercial assay or kit | Ingenio Electroporation solution | Mirus Bio LLC | # MIR 20114 | |
| Commercial assay or kit | ATPLite kit | Perkin Elmer | # 6016943 | |
| Commercial assay or kit | MycoAlert plus Mycoplasma Detection kit | Lonza | #LT07-703 | |
| Chemical compound, drug | 2-Deoxy-D-glucose | Chem-Impex Int’l, INC | # 21916 | |
| Chemical compound, drug | 2,5-Di-(t-butyl)−1,4-hydroquinone (BHQ) | SIGMA | # 112976–25G | |
| Chemical compound, drug | 2-NBDG | Cayman Chemical | # 11046 | |
| Chemical compound, drug | 3-Bromopyruvate | Aldrich Chemistry | # 16490–10G | |
| Chemical compound, drug | Cyclopiazonic acid | Alfa Aesar | # J61594 | |
| Chemical compound, drug | FCCP | SIGMA | # C2920-10MG | |
| Chemical compound, drug | Iodoacetamide | SIGMA | # I1149-5G | |
| Chemical compound, drug | Ionomycin | AdipoGen | # AG-CN2-0418 | |
| Chemical compound, drug | Oligomycin A | Alfa Aesar | # J60211 | |
| Chemical compound, drug | Rotenone | SIGMA | # R-8875–1G | |
| Chemical compound, drug | SAHA | Tocris | # 4652 | |
| Chemical compound, drug | Thapsigargin | SIGMA | # T9033-5MG | |
| Chemical compound, drug | Tunicamycin | Santa Cruz Biotechnology | # sc-3506 | |
| Software, algorithm | GraphPad Prism 7 | GraphPad Software | Version: 7 | |
| Software, algorithm | FlowJo 10 | FlowJo LLC | Version: 10.2 | |
| Software, algorithm | Adobe Illustrator | Adobe | Version: CS 5.1 |
Additional files
-
Source data 1
Data sets for main figures.
- https://doi.org/10.7554/eLife.49682.024
-
Supplementary file 1
Summary table of reporters used in CHO cells and their intended specificity.
- https://doi.org/10.7554/eLife.49682.025
-
Supplementary file 2
Free Ca2+ concentration estimates for CaCl2 containing respiration buffers.
- https://doi.org/10.7554/eLife.49682.026
-
Transparent reporting form
- https://doi.org/10.7554/eLife.49682.027
