A critical role of VMP1 in lipoprotein secretion
- University of Tokyo, Japan
- Chinese Academy of Sciences, China
- University of Massachusetts Medical School, United States
- Tokyo Medical and Dental University, Japan
Figures
Figure 1
Loss of vmp1 in zebrafish causes lethality around 9 days post fertilization and defective autophagy.
(A) Schematic representation of the Cas9-gRNA-targeted site in the zebrafish vmp1 genomic locus. The protospacer-adjacent motif (PAM) sequence is shown in red. The targeted site is underlined. A 7 …
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Figure 1—source data 1
Related to Figure 1C.
- https://doi.org/10.7554/eLife.48834.003
Figure 2 with 1 supplement
Loss of vmp1 in zebrafish causes accumulation of neutral lipids in the intestine and liver.
(A) Whole-mount oil red O staining of 8.5-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of three independent experiments. (B) Oil red O and hematoxylin staining of 6-dpf vmp1+/- and vmp1…
Figure 2—figure supplement 1
Large lipid-containing structures are not observed in the brain and skeletal muscle of vmp1-/- zebrafish or in the intestine of rb1cc1-/- and atg5-/- zebrafish.
(A) Oil red O and hematoxylin staining of 6-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of two independent experiments. Scale bars, 100 μm. (B) and (C) Transmission electron …
Figure 3 with 1 supplement
Systemic and intestinal epithelial cell-specific deletion of Vmp1 in mice causes accumulation of neutral lipids.
(A) Genotypes of offspring from Vmp1gt/+ intercross. (B) 7.5-dpc embryos were extracted from the conceptus and stained with anti-p62 antibody. Data are representative of two independent experiments. …
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Figure 3—source data 1
Related to Figure 3D,G.
- https://doi.org/10.7554/eLife.48834.008
Figure 3—figure supplement 1
Genetic map of the gene-trap and floxed alleles of the mouse Vmp1 gene in Vmp1gt mice and Vmp1flox mice, respectively.
(A) Genomic map of the wild-type and gene-trap (gt) alleles of the mouse Vmp1 gene. Black boxes indicate exons 2 to 5 and the lacZ and neo cassettes. Three loxP sites (triangles) and three primers …
Figure 4 with 1 supplement
VMP1 is important for secretion of lipoproteins.
(A and B) HepG2 cells were treated with siRNA against luciferase (Luc) or VMP1 and cultured in serum-free medium for 24 hr. Triglycerides (A) and cholesterols (B) were extracted from culture medium …
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Figure 4—source data 1
Related to Figure 4A–C.
- https://doi.org/10.7554/eLife.48834.012
Figure 4—figure supplement 1
VMP1 is required for secretion and homeostasis of lipoproteins but not for formation of cartilage structures in the zebrafish head skeleton.
(A and B) HepG2 cells were treated with siRNA against luciferase (Luc) or VMP1 and cultured in 0.1% bovine serum albumin (BSA) containing media with or without 200 μM oleic acid for 2 days. …
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Figure 4—figure supplement 1—source data 1
Related to Figure 4—figure supplement 1A–C.
- https://doi.org/10.7554/eLife.48834.011
Figure 5
Vmp1-deficient zebrafish and mice show accumulation of lipoproteins in the intestine, liver, and visceral endoderm.
Immunohistochemistry of the intestine and liver from 6-dpf vmp1+/- and vmp1-/- zebrafish (A and C) and the visceral endoderm from 7.5-dpc Vmp1gt/+ and Vmp1gt/gt mice (B and D) using anti-SEC61B …
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Figure 5—source data 1
Related to Figure 5A–D.
- https://doi.org/10.7554/eLife.48834.014
Figure 6 with 2 supplements
Depletion of VMP1 in HepG2 cells causes accumulation of abnormal lipoproteins.
(A–C) HepG2 cells were treated with siRNA oligonucleotides against luciferase (Luc) or VMP1, cultured in regular medium in the presence or absence of 200 nM oleic acid for 24 hr, and stained with …
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Figure 6—source data 1
Related to Figure 6B,C.
- https://doi.org/10.7554/eLife.48834.020
Figure 6—figure supplement 1
Depletion of VMP1 in HepG2 cells does not affect proteasome activity, ER stress, and MTTP expression.
(A) HepG2 cells were treated with siRNA oligonucleotides against luciferase (Luc) or VMP1 and cultured in regular medium in the presence or absence of 5 μM lactacystin for 24 hr. Chymotrypsin-like …
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Figure 6—figure supplement 1—source data 1
Related to Figure 6—figure supplement 1A.
- https://doi.org/10.7554/eLife.48834.017
Figure 6—figure supplement 2
APOB- and ADRP-double positive structures are not formed by proteasome inhibition, ER stress induction, MTTP inhibition, or depletion of FITM2.
(A) HepG2 cells were treated with 10 μM MG132 or 5 μM lactacystin for 24 hr (without oleic acid), and stained with BODIPY-C12 558/568 for 1 hr. Cells were fixed and stained with anti-APOB and …
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Figure 6—figure supplement 2—source data 1
Related to Figure 6—figure supplement 2A,D,E.
- https://doi.org/10.7554/eLife.48834.019
Figure 7
Neutral lipids accumulate within the ER membrane in the absence of VMP1.
(A–C) Transmission electron microscopy of intestinal epithelial cells (A) and hepatocytes (B) from 6-dpf vmp1+/- and vmp1-/- zebrafish and VMP1-depleted HepG2 cells (C). Black and white arrowheads …
Author response image 1
Interaction of VMP1 with APOB not MTTP is detected using the detergent 1% Triton-X100, but not dodecyl maltoside (1%)/cholesteryl hemisuccinate (0.2%).
HepG2 cells were transfected with FLAG-VMP1 and lysed using lysis buffer containing 1% Triton-X100 (20 mM Tris-HCI, pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton-X100) or lysis buffer containing …
Author response image 2
Immunohistochemistry of the liver from 6-dpf vmp1-/- zebrafish using anti-Sec61B antibody and LipidTOX Red.
Arrows indicate the regions where the Sec61B signals were weak. Magnified pictures of Figure 5A.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. rerio) | vmp1 | this paper | ||
| Genetic reagent (D. rerio) | rb1cc1/fip200 | PMID: 27818143 | ||
| Genetic reagent (D. rerio) | atg5 | this paper | ||
| Genetic reagent (M. musculus) | Vmp1-/- | KOMP Repository | MGI allele Vmp1tm1a(KOMP)Wtsi, clone EPD0846_3_F07 | |
| Genetic reagent (M. musculus) | Vmp1flox/flox | The European Mouse Mutant Archive | EMMA ID: EM05506 | |
| Genetic reagent (M. musculus) | Villin-Cre | Model Animal Research Center of Nanjing University | ||
| Cell line (H. sapiens) | HepG2 | ATCC | Cat. # HB-8065 RRID: CVCL_0027 | Negative for mycoplasma |
| Antibody | anti-ADRP (rabbit polyclonal) | Proteintech | Cat. #15294–1-AP | IF (1:200) |
| Antibody | anti-albumin (rabbit polyclonal) | Proteintech | Cat. #16475–1-AP, RRID: AB_2242567 | WB (1:1000) |
| Antibody | anti-APOA-I (mouse monoclonal) | Proteintech | Cat. #66206–1-Ig | WB (1:1000) |
| Antibody | anti-APOA-I (rabbit polyclonal) | Abcam | Cat. #ab64308 | IF (1:200) |
| Antibody | anti-APOB (goat polyclonal) | Rockland Immunochemicals Inc | Cat. #600-101-111, RRID: AB_2056958 | WB (1:1000) IF (1:200) |
| Antibody | anti-APOB (rabbit polyclonal) | Abcam | Cat. #ab20737, RRID: AB_2056954 | IHC (1:200) |
| Antibody | anti-APOE (mouse monoclonal) | Proteintech | Cat. #66830–1-Ig | WB (1:1000) IF (1:200) |
| Antibody | anti-α-tubulin (mouse monoclonal) | Sigma-Aldrich | Cat. #T9026, RRID: AB_477593 | WB (1:1000) |
| Antibody | anti-β-actin (mouse monoclonal) | Sigma-Aldrich | Cat. #A2228, RRID: AB_476697 | WB (1:1000) |
| Antibody | anti-BiP (rabbit polyclonal) | Abcam | Cat. #ab21685, RRID: AB_2119834 | WB (1:1000) |
| Antibody | anti-HERP (mouse monoclonal) | Chondrex | Cat. #7039 | WB (1:1000) |
| Antibody | anti-LC3 (mouse monoclonal) | Cosmo Bio | Cat. #CTB-LC3-2-IC | WB (1:1000) |
| Antibody | anti-LDH (rabbit monoclonal) | Abcam | Cat. #ab52488, RRID: AB_2134961 | WB (1:1000) |
| Antibody | anti-MTTP (mouse monoclonal) | Santa Cruz | Cat. #sc-135994, RRID: AB_2148288 | WB (1:1000) |
| Antibody | anti-p62 (rabbit polyclonal) | MBL International | Cat. #PM045, RRID: AB_1279301 | IHC (1:200) |
| Antibody | anti-PDI (mouse monoclonal) | Enzo Life Sciences | Cat. #ADI-SPA-891, RRID: AB_10615355 | WB (1:1000) |
| Antibody | anti-SEC61B (rabbit polyclonal) | Proteintech | Cat. #15087–1-AP, RRID: AB_2186411 | IHC (1:200) |
| Antibody | anti-VMP1 (rabbit polyclonal) | MBL International | Cat. #PM072 | WB (1:1000) |
| commercial assay or kit | Cholesterol Quantitation Kit | Biovision inc | Cat. #K603-100 | |
| commercial assay or kit | Cell-Based Proteasome-Glo Assays | Promega | Cat. #G8660 | |
| commercial assay or kit | Triglyceride Quantification Kit | Biovision inc | Cat. #K622-100 | |
| chemical compound, drug | BSA-conjugated oleic acid | Nacalai Tesque | Cat. #25630 | |
| chemical compound, drug | CP-346086 | Sigma-Aldrich | Cat. #PZ0103 | |
| chemical compound, drug | Lactacystin | Peptide Institute Inc | Cat. #4368-v | |
| chemical compound, drug | MG132 | Sigma-Aldrich | Cat. #M8699 | |
| chemical compound, drug | Thapsigargin | Sigma-Aldrich | Cat. #586005 | |
| chemical compound, drug | Tunicamycin | Sigma-Aldrich | Cat. #T7765 | |
| Other | BODIPY 558/568 C12 | Thermo Fisher Scientific | Cat. #D3835 | |
| Other | 4',6-diamidino-2-phenylindole (DAPI) | Sigma-Aldrich | Cat. #D9542 | |
| Other | Hoechst33342 | Dojindo Molecular Technologies | Cat. #H342 | |
| Other | LipidTOX Red | Thermo Fisher Scientific | Cat. #H34476 | |
| Other | Nile red | Thermo Fisher Scientific | Cat. #N1142 | |
| Other | Oil red O | Sigma-Aldrich | Cat. #O0625 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.48834.022
