Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 5;219(10):e202006111.
doi: 10.1083/jcb.202006111.

FIT2 is an acyl-coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis

Michel Becuwe  1   2   3 , Laura M Bond  1   2   3 , Antonio F M Pinto  4 , Sebastian Boland  1   2   3 , Niklas Mejhert  1   2   3 , Shane D Elliott  1   2   3   5 , Marcelo Cicconet  6 , Morven M Graham  7 , Xinran N Liu  7 , Olga Ilkayeva  8 , Alan Saghatelian  4 , Tobias C Walther  1   2   3   5 , Robert V Farese Jr.  1   2   3
Affiliations

FIT2 is an acyl-coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis

Michel Becuwe et al. J Cell Biol. .

Abstract

The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl 4'-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl-CoA levels as key to ER homeostasis.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
FIT2 overexpression in microsomes increases levels of oleoyl 4′-phosphopantetheine and oleoyl pantetheine upon oleoyl-CoA incubation. (A) FIT2 shares conserved motifs with LPP enzymes. C1, C2, and C3 motifs were aligned using human and yeast LPP members. FIT2 and its yeast homologues Scs3 and Yft2 have two conserved LPP motifs, C2 and C3. The conserved catalytic histidines are highlighted in red. (B) C2 and C3 catalytic histidines (highlighted in red) are evolutionarily conserved within FIT2 sequences. (C) On the basis of topology analysis (Gross et al., 2010), FIT2 N- and C-termini are predicted to be cytosolic, and the C2 and C3 motifs are predicted to be oriented toward the ER lumen. Transmembrane domains are represented as gray bars. Red boxes represent the C2 and C3 catalytic histidines. (D and E) Overexpression of FIT2 yields the production of lipids 1 and 2 upon oleoyl-CoA loading. Purified microsomes from human GnTI− 293 cells overexpressing FIT2 WT or mutated for C2 and C3 histidine to alanine (H155A and H214A, HHAA) were loaded with 25 µM mixed unlabeled and radiolabeled [14C]oleoyl-CoA, and lipids were extracted at different time points. Oleoyl-CoA was added a second time after 60 min for 10 min. Lipids were separated on a silica plate by TLC using solvents to separate polar (D) or neutral lipids (E). Autoradiograph of one representative TLC for each type of solvent is shown. OA, oleoyl coenzyme A; FA, free oleic acid; MAG, monoacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine. (F–I) Identification of lipids 1 and 2 by MS. Lipids from a microsomal extract overexpressing either FIT2 WT or FIT2 HHAA loaded with oleoyl-CoA for 10 and 30 min were analyzed by MS. Fragmentation pattern of ions 621.335 D and 525.371 D, corresponding to (F) oleoyl 4′-phosphopantetheine and (H) oleoyl pantetheine, respectively, as well as the structures assigned for each main peak, are presented. Bar graphs showing intensity level of these metabolites (G and I) found in microsomes overexpressing FIT2 WT or FIT2 HHAA at two different time points after oleoyl-CoA loading (10 and 30 min) are presented. Mean values ± SD, two-way ANOVA with Sidak test. *, P < 0.0001.
Figure S1.
Figure S1.
FIT2 homology with LPP enzymes and characterization of FIT2 activity. (A) FIT2 and its yeast homologue Scs3 share homology with LPP enzymes. FIT2 overall topology (six transmembrane domains) and the positions of C2 and C3 histidines are similar to those of other LPP enzymes (Lpp1, Lpp2, and Lpp3). Also shown are Scs3, its homologue Yft2, and some yeast LPP members (Dpp1 and Lpp1). (B) Western blotting of protein extracts from microsomal fractions purified from GnTI− 293 cells overexpressing FIT2 WT or FIT2 HHAA. (C) MS analysis of FIT2 products using palmitoyl-CoA as a substrate. Identification of lipids 1 and 2 by MS in microsomal extracts overexpressing either FIT2 WT or FIT2 HHAA loaded with palmitoyl-CoA. MS2 fragmentation patterns of ions 595.318 D and 499.356 D corresponding to palmitoyl 4′-phosphopantetheine (upper panel) and palmitoyl pantetheine (lower panel), respectively, as well as the structures assigned for each main peak, are presented. Bar graphs showing intensity level of these metabolites found in microsomes overexpressing FIT2 WT or FIT2 HHAA at 10 and 30 min after palmitoyl-CoA loading are presented (mean values ± SD; two-way ANOVA with Sidak test; *, P < 0.0001). (D) Western blot of purified FIT2 WT and HHAA proteins produced from GnTI− 293 cells. (E) Oleoyl pantetheine is produced by an enzyme independent of FIT2. Schematic representation of the experiment is shown at left. Recombinant FIT2 WT was incubated for 60 min with mixed unlabeled and radiolabeled [14C]oleoyl-CoA, and then microsomes purified from FIT2 WT or FIT2-KO SUM159 cells were added and incubated for the indicated time. As a control to visualize oleoyl pantetheine in lane 1, purified microsomes from human GnTI− 293 cells overexpressing FIT2 WT were incubated with 25 µM mixed unlabeled and radiolabeled [14C]oleoyl-CoA for 10 min. Lipids were separated by TLC with polar lipid solvent. Oleoyl-4'PP: oleoyl 4′-phosphopantetheine.
Figure 2.
Figure 2.
Recombinant human FIT2 catalyzes the cleavage of oleoyl-CoA to oleoyl 4′-phosphopantetheine but not oleoyl pantetheine. (A) Coomassie blue–stained SDS-PAGE analysis of recombinant FIT2 WT and HHAA protein purified from GnTI− 293 cells. FIT2 protein forms multimers, as confirmed by Western blot analysis (Fig. S1 D). (B) Incubation of oleoyl-CoA with FIT2 WT protein produces lipid 1. 25 ng of recombinant FIT2 WT or HHAA was incubated for the indicated time with 25 µM mixed unlabeled and radiolabeled [14C]oleoyl-CoA, and lipids produced during the reaction were analyzed by TLC using the solvent for polar lipids. A more contrasted image of the TLC is also shown. (C–E) Similar assay as that in B with FIT2 WT protein was performed with unlabeled oleoyl-CoA, and the silica plate was scraped at the location of lipid 1 (C). Reactions with FIT2 HHAA protein (D) or an empty silica lane (E) were used as controls. After extraction, lipids were directly infused into a mass spectrometer, and measured spectra are presented. (F) Fragmentation pattern after higher-energy collision dissociation of the ion 623.3499 D identified in C. (G) Incubation of oleoyl-CoA with FIT2 WT protein produces oleoyl 4′-phosphopantetheine but not oleoyl pantetheine. Schematic representation of the experiment is shown on the left. Recombinant FIT2 WT or HHAA was incubated for 60 min with mixed unlabeled and radiolabeled [14C]oleoyl-CoA, and then microsomes purified from WT GnTI− 293 cells were added and incubated for the indicated time. Lipids were separated by TLC with polar lipid solvent. Oleoyl 4′-PP, oleoyl 4′-phosphopantetheine.
Figure 3.
Figure 3.
Characterization of acyl-CoA diphosphatase activity of human FIT2. (A–C) FIT2 has oleoyl-CoA diphosphatase activity. Oleoyl-CoA diphosphatase activity was measured by the formation of oleoyl 4′-phosphopantetheine, analyzed by TLC. (A) Substrate-dependent oleoyl-CoA diphosphatase assay. The indicated concentration of mixed unlabeled and radiolabeled [14C]oleoyl-CoA was incubated for 10 min with 25 ng of purified FIT2 WT or FIT2 HHAA. The amount of oleoyl 4′-phosphopantetheine produced for each oleoyl-CoA concentration was quantified from triplicate experiments, and mean values ± SD were plotted against the oleoyl-CoA concentration (two-way ANOVA with Sidak test; *, P < 0.0001). (B) Same data as in A plotted as a Lineweaver-Burk diagram with Vmax and Km. (C) Time-dependent oleoyl-CoA diphosphatase assay. 5 µM of mixed unlabeled and radiolabeled [14C]oleoyl-CoA was incubated with 10 ng of WT FIT2 for the indicated time. The amounts of oleoyl 4′-phosphopantetheine produced at each time point were quantified, and mean values ± SD were plotted over time (two-way ANOVA with Sidak test; *, P < 0.0001). (D) Oleoyl-CoA is FIT2’s preferential substrate. 25 ng of purified FIT2 WT protein were incubated for 10 min with a mix of unlabeled and radiolabeled [14C]oleoyl-CoA (5 µM) and unlabeled CoA competitor (25 µM for ratio of 1:5 or 50 µM for ratio of 1:10). FIT2 activity was calculated relative to its activity with radiolabeled oleoyl-CoA alone. Mean values ± SD from triplicate measurements are presented in a bar graph (two-way ANOVA with Tukey test; **, P < 0.0001; *, P < 0.05). (E) Oleoyl-CoA and arachidonoyl-CoA levels are increased in FIT2-KO cells. Levels of CoA, acetyl-CoA, oleoyl-CoA, and arachidonoyl-CoA in total cell lysates from FIT2 WT and FIT2-KO cells pretreated with oleic acid for 90 min (mean values ± SD, t test; *, P < 0.01). (F) FIT2 cleaves the oleoyl-CoA phosphoanhydride bond (red arrow) to produce oleoyl 4′-phosphopantetheine and 3′-5′-ADP. Another microsomal enzyme hydrolyzes oleoyl 4′-phosphopantetheine to yield oleoyl pantetheine. Chemical structures are presented.
Figure S2.
Figure S2.
Characterization of FIT2-KO SUM159 clones. (A) Sequence of genome-edited region in FIT2-KO clones 1 and 2. In FIT2-KO clone 1, a 17-bp deletion at the end of exon 1 in the FIT2 locus yields a frame shift. In FIT2-KO clone 2, an 11-bp deletion at the end of exon 1 in FIT2 locus yields a frame shift. In the other allele, a 3-bp deletion leads to deletion of an asparagine at position 57. The gRNA position in the WT genome is indicated. (B) FIT2 mRNA levels in FIT2 WT and FIT2-KO clones as determined by qPCR. Values represent mean ± SD relative to WT cells; n = 3 (two-way ANOVA with Sidak test; *, P < 0.001). (C) No detectable FIT2 protein was observed for either FIT2-KO clone. Western blot of FIT2 WT, FIT2-KO clone 1, and FIT2-KO clone 2 crude extracts examined with anti-FIT2 antibody. (D) ER morphology was affected in FIT2-KO clone 2. Confocal images of FIT2 WT and FIT2-KO clone 2 cells transiently expressing the ER marker ssBFP-KDEL or GFP-Sec61β. White arrows indicate ER aberrations. Scale bar = 10 µm. (E) FIT2-KO clone 2 cells form fewer and smaller LDs. Confocal images of FIT2 WT and FIT2-KO clone 2 cells treated with 500 µM oleic acid for 48 h. LDs were stained with BODIPY. Representative confocal images are presented. Scale bar = 10 µm. (F) FIT2-KO clone 2 displayed an alteration of lipid synthesis enzyme expression. mRNA levels of genes involved in the Kennedy pathway were assessed by qPCR analysis of FIT2 WT and FIT2-KO clone 2 cells. Values represent mean ± SD relative to WT cell level; n = 3 (two-way ANOVA with Sidak test; *, P < 0.05). (G) FIT2-KO clone 2 displayed a defect in TG synthesis. FIT2 WT and FIT2-KO clone 2 cells were pulse labeled with [14C]oleic acid, and incorporation into TG was measured over time by TLC. Mean value of TG band intensity was plotted over time and quantified from three independent measurements. Values were calculated relative to the value of WT cells at 24 h (two-way ANOVA with Sidak test; *, P < 0.01). The TG synthesis rate of FIT2-KO clone 1 is also shown for comparison.
Figure 4.
Figure 4.
FIT2-deficient human SUM159 cells have altered ER morphology. (A) Confocal images of FIT2 WT and FIT2-KO SUM159 cells transiently expressing the ER marker ssBFP-KDEL or GFP-Sec61β. White arrows indicate ER aberrations. Scale bar = 10 µm. (B and C) Representative thin-section EM scans from FIT2-KO cells showing (B) ER whorls highlighted with black arrows (scale bars = 500 nm) and (C) ER dilation highlighted with black arrows (scale bars = 1 µm [top image], 500 nm [bottom image]). (D) ER whorls in FIT2-deficient cells are localized perinuclearly. Representative lattice light-sheet microscopy images of FIT2 WT and FIT2-KO cells transiently expressing oxGFP-KDEL. ER abnormalities are indicated with white arrows. Scale bar = 10 µm. A rendering image shows the ER meshwork (brown) and the perinuclear localization of ER whorls (yellow shapes). Scale bar = 4 µm. (E) The ER stress marker XBP1 is normally regulated in FIT2-KO cells. XBP1 mRNA from FIT2 WT or FIT2-KO cells, treated for the indicated times with 100 µM palmitate, was amplified by qPCR and loaded onto an agarose gel. Unspliced XBP1 (Xbp1u) was observed as a 152-bp band, and the spliced form (XBP1s) was observed as a 126-bp band. (F) Several ER stress markers are elevated in FIT2-KO cells. qPCR analysis of the indicated genes was performed on FIT2 WT and FIT2-KO cells untreated or treated with 100 µM palmitate for 16 h. Values represent mean ± SD relative to WT value; n = 3 (two-way ANOVA with Sidak test; *, P < 0.05). (G) Western blot analysis of the indicated proteins was performed on FIT2 WT and FIT2-KO cells untreated or treated with 100 µM palmitate for 16 h or tunicamycin (4 µg/ml) for 14 h. Quantification of band intensity relative to untreated WT controls is shown under each panel. (H and I) FIT2 siRNA-treated cells have more ER sheets than control siRNA-treated cells. (H) Confocal images of WT cells transiently expressing the ER marker oxGFP-KDEL treated with control or FIT2 siRNA for 72 h. An inlay highlights the higher proportion of sheetlike structures at the cell periphery in FIT2 siRNA-treated cells than in controls. Scale bar = 10 µm. (I) Quantification of ER features (sheets and tubules) relative to the cell volume in cells treated with control siRNA or FIT2 siRNA for 3 d and transiently expressing the ER marker oxGFP-KDEL. Quantification was performed as described in the Materials and methods section (two-way ANOVA with Bonferroni test; *, P < 0.0001). (J) Knock-down of FIT2 does not induce ER stress. mRNA levels of the indicated genes were assessed by qPCR analysis of WT cells pretreated with control or FIT2 siRNA for 72 h. Values represent mean ± SD relative to WT value; n = 3. The figure is representative of two independent experiments (two-way ANOVA with Sidak test; *, P < 0.05).
Figure S3.
Figure S3.
Impact of FIT2 deletion on other organelles. (A) Representative confocal images of immunofluorescence using antibodies targeting markers of the ER (anti–protein disulfide isomerase) and autophagy (anti-LC3). Scale bar = 10 µm. (B) Representative confocal images of FIT2 WT and FIT2-KO cells cotransfected with RTN4a-GFP and ssBFP-KDEL. White arrows indicate the presence of ER whorls. Scale bar = 5 µm. (C) Representative confocal images of immunofluorescence using antibodies targeting markers of the Golgi apparatus (anti-GM130), peroxisome (anti-catalase), and lysosome (anti-LAMP1) in FIT2 WT and FIT2-KO SUM159 cells. Scale bar = 10 µm.
Figure S4.
Figure S4.
FIT2-KD efficiency, ER feature quantification, and characterization of the cell line expressing endogenously GFP-tagged FIT2. (A) Cells treated with FIT2 siRNA for 72 h reduced FIT2 protein levels by 80%. Cells were treated with control or FIT2 siRNA for the indicated time, and FIT2-KD efficiency was determined by Western blotting with an anti-FIT2 antibody. Tubulin was used as a loading control. Quantification of band intensities relative to control siRNA at 24 h is shown under the tubulin panel. (B) FIT2 protein abundance based on Western blots in A was plotted relative to control siRNA for each time point. (C) Segmentation of ER features by machine learning. Cells treated with control siRNA for 72 h transiently expressing the ER marker ER-oxGFP were imaged. One confocal image of a Z-stack is presented (original image). As detailed in the Materials and methods section, machine learning was used to segment the ER features (sheets, tubules, and outside of the cell/cytosol). Scale bar = 10 µm. (D) Western blot analysis of the CRISPR-engineered GFP knock-in cell line using anti-FIT2 antibody. FIT2 WT and FIT2-KO cell extracts are used as controls. (E) Western blot using anti-FIT2 antibody on crude extracts from WT cells, FIT2-KO cells, and FIT2-KO cells expressing GFP-FIT2 WT or H214A under PGK promoter stably integrated in the safe harbor AAVS1 genomic locus. Endogenous FIT2 was observed at the expected size (22 kD) only in the WT cell extract. GFP-tagged forms of FIT2 were observed at the expected size (45 kD) in WT GFP-FIT2 and H1214A stable cell lines. Ponceau staining was used to control for protein loading.
Figure 5.
Figure 5.
FIT2 is required for normal LD formation. (A) FIT2 localizes diffusely throughout the ER. SUM159 cells expressing endogenous GFP-tagged FIT2 transfected with plasmid expressing the luminal ER marker ssBFP-KDEL were imaged before and after addition of oleic acid at the indicated time. LDs were stained with LipidTOX. Scale bar = 10 µm. (B) FIT2 deletion results in fewer and smaller LDs formed in response to oleate treatment. Time course of LD formation in FIT2 WT and FIT2-KO SUM159 cells. Cells were treated with 500 µM oleic acid for the indicated times, and LDs were stained with BODIPY 493/503. Scale bar = 10 µm. (C) Quantification of LD formation shown in B. n = 4 cells. Values represent mean ± SD (two-way ANOVA; *, P < 0.001). (D) Reduced triacylglycerol synthesis in FIT2-KO cells. Cells were pulse-labeled with [14C]oleate, and incorporation into TG was measured over time by TLC. Mean value of TG band intensity was plotted over time and quantified from three independent measurements ± SD. Values were calculated relative to WT cells’ highest value at 360 min (two-way ANOVA with Sidak test; *, P < 0.0001). (E) FIT2-deficient cells form fewer LDs for similar amounts of synthesized TG. Average LD number per cell measured in B was plotted against TG amount calculated in C. (F) Reduced mRNA expression of lipid synthesis enzymes in FIT2-KO cells. mRNA levels of genes involved in the Kennedy pathway in FIT2 WT and FIT2-KO cells were assessed by qPCR analysis. Values represent mean ± SD relative to WT value; n = 3. The figure is representative of three independent experiments (two-way ANOVA with Sidak test; *, P < 0.01). (G) Knockdown of FIT2 results in reduced DGAT1 and LIPIN1 mRNA expression levels. mRNA levels of genes involved in the Kennedy pathway were assessed by qPCR analysis of WT cells pretreated with control or FIT2 siRNA-treated cells. Values represent mean ± SD relative to WT value; n = 3. The figure is representative of two independent experiments (two-way ANOVA with Sidak test; *, P < 0.05). (H) FIT2 siRNA-treated cells have fewer LDs than control siRNA-treated cells. WT cells were pretreated for 48 h with siRNA targeting FIT2 or control siRNA, and then oleic acid was added for another 48 h before imaging. Scale bar = 5 µm.
Figure S5.
Figure S5.
Yeast assays of FIT2 and SCS3 function. (A) Representative confocal images of inducible lipid droplet (iLD) strain deleted for SCS3 and YFT2 (iLD scs3Δ yft2Δ) or not (iLD) after overnight growth in raffinose (repressed LD condition) and after galactose addition (induced LD condition) at the indicated times. LDs were stained with BODIPY 493/503. Scale bar = 5 µm. (B) Cells were classified depending on their number of LDs over time (from 0 to 7 LDs per cell). Results are presented as a percentage of cells for each time point after galactose addition (n = 100 to 150 cells). (C) N- and C-terminally GFP-tagged Scs3 orthologues are functional, but only N-terminally GFP-tagged FIT2 is functional. N- and C-terminally GFP-tagged versions of Scs3 and FIT2 expressed from plasmids in scs3Δ cells were grown on complete (+INO−CHO) or inositol-deprived medium (−INO+CHO). (D and E) Scs3 LPP mutants do not have dominant-negative effects. (D) WT or LPP mutant forms of Scs3 expressed from plasmid in WT cells were grown on complete (+INO-CHO) or inositol-deprived medium (−INO+CHO) for 2 d, and (E) their subcellular localizations were assessed by confocal fluorescence microscopy. Localization of the same constructs in scs3Δ cells is also presented. ER whorls are indicated by white arrows. Scale bar = 5 µm.
Figure 6.
Figure 6.
Catalytic residues of FIT2 and Scs3 are required for their Function in vivo in yeast. (A) C2 or C3 histidine mutations of Scs3 or FIT2 did not rescue scs3Δ inositol auxotrophy. WT cells, scs3Δ cells, and scs3Δ cells expressing GFP-tagged Scs3 or FIT2 WT or mutated for C2 histidine to alanine (H235A for Scs3, H155A for FIT2) or C3 histidine to alanine (H350A for Scs3, H214A for FIT2) on a plasmid were serially diluted and spotted on complete solid medium (+INO−CHO) or inositol-depleted (−INO+CHO). To aggravate the growth defect (Villa-García et al., 2011), inositol-deprived medium was supplemented with choline (+CHO), and cells were grown at 37°C. The plates were imaged after 4–5 d. (B) SCS3 deletion leads to formation of ER patches. Maximum-intensity projections of representative deconvolved confocal image stacks from WT and scs3Δ cells expressing the ER marker ssRFP-HDEL are shown; white arrows highlight ER patches. Scale bar = 2 µm. (C) Reintroduction of Scs3-GFP mutated at C2 or C3 histidine does not restore normal ER shape in scs3Δ cells. Subcellular localization of Scs3-GFP WT or H235A and H350A, respectively, were assessed in scs3Δ cells. Maximum-intensity projections of representative deconvolved confocal image stacks are presented. Impact on ER shape was addressed with ssRFP-HDEL marker; white arrows highlight ER patches. Scale bar = 2 µm. (D) Reintroduction of GFP-FIT2 mutated at C2 or C3 histidine did not restore normal ER shape in scs3Δ. Subcellular localization of GFP-FIT2 WT or H155A and H214A, respectively, were assessed in scs3Δ cells. Maximum-intensity projections of representative deconvolved confocal image stacks are presented. Impact on the ER shape was addressed with ssRFP-HDEL marker; white arrows highlight ER patches. Scale bar = 2 µm.
Figure 7.
Figure 7.
FIT2 catalytic residues are required for ER homeostasis and normal lipid storage in human cells. (A and B) Reintroduction of GFP-FIT2 mutated at conserved histidines did not restore normal ER shape in FIT2 KO cells. (A) Representative confocal images of FIT2 WT cells and FIT2-KO cells transiently coexpressing the ER marker ssBFP-KDEL and GFP-FIT2 WT or C2 (H155A) or C3 histidine mutants (H214A). White arrows highlight ER patches. Scale bar = 10 µm. (B) Quantification of the percentage of rescued ER shape (n = 50) is presented under the images. ND, not detected. (C and D) Reintroduction of GFP-FIT2 mutated at H214A did not restore normal ER stress markers expression. (C) qPCR analysis of the indicated genes (values represent mean ± SD; n = 3, representative of three independent experiments; two-way ANOVA with Sidak test; *, P < 0.01; **, P < 0.001). (D) Western blot analysis of ER stress proteins in FIT2 WT cells, FIT2-KO cells, and FIT2-KO cells stably expressing either WT GFP-FIT2 (KO + WT) or GFP-FIT2-H214A (KO + HA). Quantification of band intensity relative to WT untreated is shown under each panel. (E) Reintroduction of WT GFP-FIT2 or H155A did not restore normal TG synthesis rate in FIT2-KO cells. FIT2 WT, FIT2-KO cells, and FIT2-KO cells stably expressing either WT GFP-FIT2 (KO+WT) or GFP-FIT2-H214A (KO+HA) were pulse labeled with [14C]oleate, and incorporation into TG was measured by TLC over time during oleate treatment. Mean value of TG band intensity was plotted over time and quantified from three independent measurements. Values were calculated relative to the value of WT cells at 24 h (two-way ANOVA with Sidak test; *, P < 0.01). (F) Reintroduction of WT GFP-FIT2 or H214A did not restore normal expression of neutral lipid synthesis genes in FIT2-KO cells. qPCR analysis of the indicated genes was performed on FIT2 WT cells, FIT2-KO cells, and add-back cell lines. Values represent mean ± SD; n = 3. The figure represents three independent experiments (two-way ANOVA with Sidak test; *, P < 0.01). (G) Reintroduction of WT GFP-FIT2 but not H214A restores LD budding in FIT2-KO cells. FIT2-KO cells were grown together with add-back cell lines expressing GFP-FIT2 WT (top panels) or GFP-FIT2 H214A (bottom panels), and 500 µM oleic acid was added for 24 h before imaging. LDs were stained with LipidTOX. Nuclei were stained with Hoechst. FIT2-KO cells (labeled with asterisks) were distinguished from add-back cells on the basis of GFP signal. Scale bar = 25 µm. MW, molecular weight.
See this image and copyright information in PMC

References

    1. Aguet F., Upadhyayula S., Gaudin R., Chou Y.-Y., Cocucci E., He K., Chen B.-C., Mosaliganti K., Pasham M., Skillern W., et al. . 2016. Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy. Mol. Biol. Cell. 27:3418–3435. 10.1091/mbc.e16-03-0164 - DOI - PMC - PubMed
    1. Becuwe M., Bond L.M., Mejhert N., Boland S., Elliott S.D., Cicconet M., Liu X.N., Graham M.M., Walther T.C., and Farese R.V. Jr. 2018. FIT2 is a lipid phosphate phosphatase crucial for endoplasmic reticulum homeostasis. bioRxiv. doi: (Preprint posted March 29, 2018)10.1101/291765 - DOI
    1. Bligh E.G., and Dyer W.J.. 1959. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 37(8):911–917. 10.1139/o59-099 - DOI - PubMed
    1. Cartwright B.R., Binns D.D., Hilton C.L., Han S., Gao Q., and Goodman J.M.. 2015. Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology. Mol. Biol. Cell. 26:726–739. - PMC - PubMed
    1. Chan T.F., and Vese L.A.. 2001. Active contours without edges. IEEE Trans. Image Process. 10:266–277. 10.1109/83.902291 - DOI - PubMed

Publication types

MeSH terms