Augmenter of liver regeneration regulates cellular iron homeostasis by modulating mitochondrial transport of ATP-binding cassette B8
- Feinberg Cardiovascular and Renal Research Institute, Northwestern University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Downregulation of Alr results in cytoplasmic Fe/S cluster maturation defects.
(A) mRNA (n = 6) and (B) protein (n = 3) levels of Alr in wild type (WT) mouse embryonic fibroblasts (MEFs) with or without Alr downregulation. GPAT protein levels (n = 3, C), xanthine oxidase (XO, …
Figure 1—figure supplement 1
Acute downregulation of Alr does not affect cell viability or mitochondrial respiration.
(A) Representative western blotting of total cell lysate, cytosolic and mitochondrial fraction from MEFs demonstrate the localization of long and short isoforms of ALR. OPA1 serves as a …
Figure 2 with 1 supplement
Downregulation of ALR increases Tfrc levels and cellular iron content through IRP1.
Tfrc mRNA levels (A) and transferrin-dependent iron uptake (B) in wild type (WT) mouse embryonic fibroblasts (MEFs) with Alr downregulation (n = 5–6). (C) Steady-state 55Fe levels in cytosolic and …
Figure 2—figure supplement 1
ALR regulates Tfrc mRNA levels through a posttranscriptional mechanism independent of IRP2.
(A) Schematic representation of the Tfrc promoter reporter construct. +1 represents transcription start site on Tfrc genomic sequence. (B) Tfrc promoter activities are measured in wild type (WT) …
Figure 3 with 1 supplement
Overexpression of full-length but not cytosolic isoform of ALR rescues the iron-sulfur cluster maturation defects from ALR downregulation.
Tfrc mRNA levels (n = 6, A) and transferrin-dependent iron uptake (n = 4–6, B) were measured in wild type (WT) mouse embryonic fibroblasts (MEFs) with endogenous Alr downregulation and concurrent …
Figure 3—figure supplement 1
Validation of isoform-specific overexpression of ALR with concurrent downregulation of endogenous Alr.
(A) Representative western blotting results showing the subcellular localization of full-length and cytosolic isoform of ALR. (B) Densitometry analysis mitochondrial localization of each construct …
Figure 4 with 1 supplement
Downregulation of ALR in HEK293 cells reduces mitochondrial ABCB8 protein level.
(A) mRNA levels of ABCB7 and ABCB8 in HEK293 cells with ALR downregulation harvested 48 hr after siRNA transfection (n = 6). Total cellular levels of ABCB7 (B) and ABCB8 (C) in HEK293 cells with ALR …
Figure 4—figure supplement 1
Downregulation of ALR in HEK293 cells results in cytosolic Fe/S cluster maturation defects.
(A) ALR mRNA levels in HEK293 cells treated with ALR siRNA and harvested 48 hr after transfection (n = 6). (B) Representative western blot showing ALR protein levels in HEK293 cells treated with ALR …
Figure 5 with 1 supplement
Mitochondrial import of ABCB8 requires ALR, MIA40, TIM23, and mitochondrial membrane potential.
Imported levels of MIA40-His (n = 6, A), whose mitochondrial translocation depends on Mia40/ALR protein transport pathway, ABCB8-His (n = 9, B), and ABCB7-His (n = 3, C) in cells treated with ALR …
Figure 5—figure supplement 1
Downregulation of ALR or MIA40 does not affect mitochondrial import of SU9-eGFP.
(A) Representative western blot showing time-dependent accumulation of GFP protein in cells stably expressing rtTA3 and transfected with doxycycline (DOXY)-inducible GFP expression plasmid. (B) …
Figure 6 with 1 supplement
ABCB8 interacts with Mia40 during its transport into mitochondria.
(A) Representative western blot image of mitochondrial lysates and His-tag pulldown fraction from cells overexpressing His-tagged MIA40 or empty vector. (B) Co-immunoprecipitation of His-tagged …
Figure 6—figure supplement 1
Individual mutation of the five conserved cysteines in ABCB8 minimally impacts its interaction with MIA40.
(A) Western blot results showing co-immunoprecipitation of His-tagged MIA40 and various mutants of Flag-tagged ABCB8 in cells overexpressing indicated constructs. (B) Clustal Omega alignment between …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit polyclonal anti-GPAT antibody | Dr. Roland Lill, University of Marburg | N/A | WB(1:1000) |
| Antibody | Rabbit polyclonal anti-ABCB8 antibody | Lab generated. For reference, see Ardehali et al 2004. | N/A | WB(1:1000) |
| Antibody | Rabbit polyclonal anti-GAPDH antibody | Abcam | ab9485 | WB(1:6000) |
| Antibody | Rabbit polyclonal anti-ALR antibody | ProteinTech | 11293–1-AP | WB(1:1000-1:4000) |
| Antibody | Rabbit polyclonal anti-ABCB7 antibody | ProteinTech | 11158–1-AP | WB(1:500) |
| Antibody | Rabbit polyclonal anti-alpha tubulin antibody | Abcam | ab4074 | WB(1:2000) |
| Antibody | Mouse monoclonal anti-SDH70 antibody (2E3GC12FB2AE2) | Invitrogen | 459200 | WB(1:7000) |
| Antibody | Mouse monoclonal anti-6x-His antibody | ProteinTech | 66005–1-Ig | WB(1:4000-1:6000) |
| Antibody | Mouse monoclonal anti-GFP antibody | ProteinTech | 66002–1-Ig | WB(1:4000-1:6000) |
| Antibody | Mouse monoclonal anti-ATP5A antibody [15H4C4] - Mitochondrial Marker | Abcam | ab14748 | WB(1:4000) |
| Antibody | Rabbit polyclonal anti-Mia40 antibody | ProteinTech | 21090–1-AP | WB(1:4000) |
| Antibody | Rabbit polyclonal anti-Tim23 antibody | ProteinTech | 11123–1-AP | WB(1:4000) |
| Antibody | Mouse monoclonal Flag M2 antibody | Sigma | F1804 | WB(1:1000-1:4000) |
| Antibody | Mouse monoclonal anti-COX4 antibody [1A12A12] | Abcam | ab110272 | WB(1:1000) |
| Antibody | Rabbit polyclonal anti-beta actin antibody | Cell Signaling | 4967 | WB(1:2000) |
| Antibody | HRP-conjugated donkey polyclonal anti-rabbit IgG antibody | Jackson ImmunoResearch | 711-035-152 | WB(1:2000-1:12000) |
| Antibody | HRP-conjugated donkey polyclonal anti-mouse IgG antibody | Jackson ImmunoResearch | 715-035-150 | WB(1:2000-1:12000) |
| Chemical compound, drug | 55Fe radionuclide. | Perkin Elmer | NEZ043002MC | |
| Chemical compound, drug | RNA-Stat60 | Teltest | Cs-502 | |
| Chemical compound, drug | Glycogen | Life Technologies | AM9510 | |
| Chemical compound, drug | Lipofectamine 2000 | Life Technologies | 11668019 | |
| Chemical compound, drug | Apo-transferrin, human | Sigma | T1147 | |
| Chemical compound, drug | Dharmafect Transfection Reagent I | Dharmacon | T-2001–03 | |
| Chemical compound, drug | Deferoxamine mesylate | Sigma | D9533-1G | (150 µM) |
| Chemical compound, drug | Doxycycline | Fisher Scientific | AC446060050 | (1 µg/mL) |
| Chemical compound, drug | Blasticidin | Life Technologies | R21001 | (10 µg/mL) |
| Chemical compound, drug | Puromycin | Sigma | P8833-25MG | (6 µg/mL) |
| Chemical compound, drug | DMEM, 4.5 g/L glucose with L-glutamine and sodium pyruvate | Corning | 10-013CV | |
| Chemical compound, drug | MEM | Corning | 15-010CV | |
| Chemical compound, drug | Sodium pyruvate solution | Corning | 25–000 CI | |
| Commercial assay, kit | qScript cDNA Synthesis Kit | Quanta | 95047–500 | |
| Commercial assay, kit | PerfeCTa SYBR Green FastMix | Quanta | 95074–05K | |
| Commercial assay, kit | Dual-Luciferase Reporter Assay System | Promega | E1980 | |
| Commercial assay, kit | BCA Protein Assay Kit | Pierce | 23225 | |
| Commercial assay, kit | Amplex Red Xanthine/Xanthine Oxidase Assay Kit | ThermoFisher | A22182 | |
| Commercial assay, kit | Complex I Enzyme Activity Microplate Assay Kit | Abcam | ab109721 | |
| Commercial assay, kit | Complex II Enzyme Activity Microplate Assay Kit | Abcam | ab109908 | |
| Commercial assay, kit | Mitochondria Isolation Kit for Cultured Cells | Thermo Scientific | 89874 | |
| Commercial assay, kit | Aconitase Activity Assay Kit | Abcam | ab109712 | |
| Commercial assay, kit | Seahorse Extracellular FluxPak | Agilent | 102416–100 | |
| Cell line (human) | HEK293 cells | ATCC | CRL-1573 | |
| Transfected construct (human) | HEK293 cells stably expressing rtTA3 | This manuscript. | N/A | Cells were stably infected with rtTA3 lentivirus made from pLenti CMV rtTA3 Blast (w756-1) plasmid |
| Cell line (mouse) | WT and IRP2 KO mouse embryonic fibroblasts | Dr. Tracey Rouault NICHD, NIH | N/A | |
| Transfected construct (mouse) | WT MEFs stably expressing Irebp1 shRNA | This manuscript | N/A | Cells were stably infected with lentivirus expressing mouse Irebp1 shRNA made from rtTA3 lentivirus made from pGIPZ-shIrebp1 |
| Sequence-based reagents | siGenome siRNA against mouse GFER | Dharmacon | MQ-065586-01-0002 | |
| Sequence-based reagents | Negative Control siRNA | Qiagen | 1027310 | |
| Sequence-based reagents | siGenome siRNA against human GFER | Dharmacon | MQ-065586-01-0002 | |
| Sequence-based reagents | siGenome siRNA against mouse IRP1 | Dharmacon | MQ-043541-01-0002 | |
| Sequence-based reagents | siRNA against mouse ALR 3’UTR | Qiagen | SI01011199, SI01011220 | |
| Sequence-based reagents | Primer for qRT-PCR (Supplementary file 1) | This manuscript | N/A | Please see Supplementary file 1 |
| Recombinant DNA reagent | pGIPZ-shIrebp1 | OpenBioSystems | V2LMM_82191 | |
| Recombinant DNA reagent | pRL-TK (Renilla Luciferase) | Promega | E2241 | Luciferase reporter construct expressing Renilla luciferase |
| Recombinant DNA reagent | pMIR-REPORT | Ambion | AM5795 | Empty 3’UTR luciferase reporter construct expressing Firefly luciferase |
| Recombinant DNA reagent | pGL3 Basic Firefly Reporter construct | Promega | E1751 | Promoter luciferase reporter construct expressing Firefly luciferase |
| Recombinant DNA reagent | Mouse Tfrc 3’-UTR luciferase and mutants | This manuscript and Bayeva et al., 2012 | N/A | |
| Recombinant DNA reagent | Mouse Tfrc promoter luciferase construct | This manuscript | N/A | |
| Recombinant DNA reagent | pBI-MCS-EGFP | Addgene | 16542 | |
| Recombinant DNA reagent | pLenti CMV rtTA3 Blast (w756-1) | Addgene | 26429 | |
| Software, algorithm | GraphPad Prism | GraphPad | Version 9 | |
| Software, algorithm | ImageJ | NIH | 1.53 c |
Additional files
-
Supplementary file 1
Primer sequences for quantitative RT PCR.
The protocol for qRT-PCR were described in 'Materials and methods' section.
- https://cdn.elifesciences.org/articles/65158/elife-65158-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/65158/elife-65158-transrepform-v2.docx
