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. 2013 Feb 25;24(4):384-99.
doi: 10.1016/j.devcel.2013.01.013. Epub 2013 Feb 14.

Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets

Florian Wilfling  1 , Huajin Wang, Joel T Haas, Natalie Krahmer, Travis J Gould, Aki Uchida, Ji-Xin Cheng, Morven Graham, Romain Christiano, Florian Fröhlich, Xinran Liu, Kimberly K Buhman, Rosalind A Coleman, Joerg Bewersdorf, Robert V Farese Jr, Tobias C Walther
Affiliations

Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets

Florian Wilfling et al. Dev Cell. .

Abstract

Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

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Figures

Figure 1
Figure 1. A Subset of TG Synthesis Isoenzymes Localizes to LDs in Drosophila S2 Cells
(A) Overview of de novo TG synthesis. CG numbers identify the Drosophila genes encoding each activity. (B) Neighbor-joining tree based on the degree of sequence similarity between acyltransferase domains of the GPAT/AGPAT family from Homo sapiens (h), Mus musculus (m), and Drosophila melanogaster (dm). The tree branch lengths are scaled in substitutions per site. (C) Abundance profiles of proteins throughout the fractions of an LD purification measured by quantitative mass spectrometry. Normalized peptide abundances measured by proteomic analyses of fractions of a purification from cells metabolically labeled with heavy isotope-containing amino acids are shown normalized to an LD protein standard obtained by a similar purification. Fraction 9 is the LD fraction. The left graph indicates profiles representative of cytosolic (GAPDH), ER (GRP78), and LD (CCT1) proteins. The right graph shows profiles of isoenzymes involved in TG synthesis highly enriched in the LD fraction. (D) GPAT4, AGPAT3, or DGAT2 tagged with fluorescent cherry tag (red) localizes to LDs (BODIPY, green) in oleate-loaded Drosophila S2 cells. (E) GPAT4 accumulates at LDs during LD formation. Cherry-GPAT4 images during LD formation induced by oleate-containing medium at the indicated time points are shown. (F) Immunofluorescence staining of GPAT4 (red) on forming LDs (BODIPY, green). Scale bars, 10 μm (overview) and 1 μm (magnification). See also Figure S1, and Tables S1 and S2.
Figure 2
Figure 2. GPAT4 Relocalizes from the ER to the Surface of LDs
(A) A time course of LD formation induced by oleate-containing medium shows segregation of cherry-tagged GPAT4 (red) from the ER marker (GFP-SEC61β; green). Optical sections focus on LDs, resulting in different sections of the nuclear envelope (NE) at each time point (plane of the NE at 0 hr; cross-section at other time points). Scale bars, 10 μm (overview) and 1 μm (magnification). (B) GPAT4 tagged with fluorescent cherry tag (red) accumulates in specialized regions of the ER (marked by GFP-SEC61β; green). Scale bars, 10 μm (overview) and 1 μm (magnification). Intensity profiles of the two signals along the dotted line are shown. Pearson correlations for GFP-Sec61β and cherry-GPAT4 signal within the white boxes at 0.5 or 3 hr of oleate are shown. (C) GPAT4 fractionates with LDs after oleate treatment. Western blots of equal protein amounts of cytosol, microsome, and LD fractions against the LD marker CCT1, the ER marker GRP78, and the cytosolic marker tubulin are shown. (D) cherry-GPAT4 relocalizes to LDs from the ER during LD formation in cells where the synthesis of new proteins is blocked by cycloheximide added prior to oleate. On the right, sizes of 40 GPAT4-containing LDs from cells cultured 8 hr in control medium or medium containing cycloheximide are shown. (E) Cherry-GPAT4 signal covers the entire LD. z sections and a 3D reconstruction of cherry-GPAT4 containing LD are shown. Scale bar, 2 μm. (F) Cherry-GPAT4 decreases uniformly during continuous bleaching of a small LD surface section (upper panels). Cherry-GPAT4 recovers uniformly after photobleaching of a small LD surface section (lower panel). Scale bars, 2 μm. (G) Endogenous GPAT4 (red) colocalizes with the LD marker CGI58 (green) but segregates from the ER (ss-GFP-KDEL; green). Scale bar, 1 μm. (H) Silver-enhanced Immunogold staining of endogenous GPAT4 on LDs in thin-section EM. Red and blue arrows show representative gold particles on the surface of LDs. Yellow arrows show close-by ER structures. Scale bar, 500 nm. See also Figures S2 and S3.
Figure 3
Figure 3. GPAT4 Is an Integral Membrane Protein Containing a Membrane Hairpin Sequence Localizing to LDs via ER-LD Bridges
(A) GPAT4 is a membrane protein not extracted from microsomes by buffers containing 1% SDS, 100 mM Na2CO3, or 1 M NaCl. (B) Similar to cytosolic ribosomal protein S6, but in contrast to GRP78, the N and C termini of GPAT4 are in the cytosol and accessible to Proteinase K in the presence or absence of Triton X-100. (C) Membrane topology model of GPAT4. Blue regions indicate predicted transmembrane domains and orange regions the catalytic domain. (D) The membrane hairpin of GPAT4 is necessary and sufficient for ER targeting. GPAT4 constructs containing (i) the entire GPAT4 sequence (ii) only the hairpin loop (iii) having replaced the hairpin loop with a flexible linker (iv) the interhelix segment of the hairpin loop with a linker sequence consisting of loop sequence of AGPAT1 were localized by fluorescence microscopy relative to an ER (GFP-SEC61β; in cells without oleate) or LD marker (BODIPY; in cells with oleate). Scale bars, 10 μm (overview) and 1 μm (magnification). (E) GPAT4 is mobile within the ER, as indicated by fast recovery of signal into a bleached region (indicated by a box). Representative images are shown. Scale bar, 10 μm. Quantitation shows the recovery of GPAT4-GFP signal from three independent experiments. Error bars indicate SDs. (F) Fluorescence recovery in a bleached region of GFP-GPAT4 signal on a forming LD (after 2 hr of incubation with oleate-containing medium). Representative images are shown. Single experiments show recovery of GFP-GPAT4 signal with different rates. Scale bar, 10 μm. Histogram of fluorescence recovery levels from 30 FRAP experiments. (G) Recovery of GPAT4 occurs through ER-LD connections. Representative images from a FRAP movie are shown. Arrowheads highlight bridges between the GPAT4 ER and LD surface signal. Scale bar, 2 μm. (H) Thin-section EM analysis of high-pressure frozen cells shows membrane continuity between LDs and the ER (highlighted by boxes). Blue arrows indicate ER-LD connections. See also Figures S4 and S5, and Movie S1.
Figure 4
Figure 4. GPAT4 Localizes to Growing LDs
(A) LDs are able to in vitro generate TG, DG, and LPA from radiolabeled oleoyl-CoA, resolved by TLC. Western blot analysis of the LD fractions (lower panel). (B) A subset of LDs grows as shown by 3D time-lapse live-cell imaging from 0 to 8 hr of oleate loading. Representative optical midsections and reconstructions for a growing (top) and nongrowing (bottom) LD are shown from 240 to 360 min. Scale bars, 1 μm (BODIPY and 3D) and 2 μm (whole cell). The graph shows LD volume over time. (C) Only LDs with GPAT4 expand over time. Scatterplots and box plots (median, lower and upper quartile, 1.5 interquartile range for whiskers) of diameters of LDs with (red) or without (black) GPAT4 are shown during their formation induced by oleate-containing medium. (D) GPAT4 (green) localizes to expanding LDs marked by CCT1 (red) by immunofluorescence. After imaging the immunofluorescence signal, cells were stained with BODIPY to visualize all LDs (green channel). Scale bars, 10 μm (overview) and 1 μm (magnification).
Figure 5
Figure 5. LD-Localized TG Synthesis Enzymes Are Required for the Formation of Large LDs
(A) Histogram of LD sizes from control RNAi-treated cells shows a bimodal distribution of LD diameters (red curve), modeled by two normal distributions ((f(x)=a1×e−(x−b1)2∕c12+a2×e−(x−b2)2∕c22; orange and green dashed lines; a1, 42.25; b1, 0.7672; c1, 0.2; a2, 8.377; b2, 1.936; c2, 0.8925) for small (gray area) and large LDs (yellow area). (B) Depletion of LD-localized TG synthesis enzymes (GPAT4, AGPAT3, DGAT2; black, light-blue, dark-green density curves) diminishes the abundance of large LDs present in the control (red line). Scale bar, 10 μm. (C) Depletion of ER-localized TG synthesis enzymes (GPAT1, AGPAT1, AGPAT2, DGAT1; orange, blue, dark-blue, light-green density curves) has little effect on the size distribution of large LDs. Representative images are shown. Scale bar, 10 μm. (D) Effect of TG synthesis enzyme depletion by RNAi on TG levels. A box plot quantitation of BODIPY signal area per cell (median, lower, and upper quartile, 1.5 interquartile range for whiskers) is shown on top. Significance was tested by ANOVA. Bottom panels show a TLC of TG, PC, and PE levels in cells depleted for the indicated enzymes. (E) Although both GPAT4 and GPAT1 transfection rescue total TG levels in GPAT4-depleted cells, only GPAT4 transfection leads to formation of large LDs. Scale bar, 10 μm. Significance was tested by ANOVA. (F) Overexpression of DGAT2 and DGAT1 leads to large or small LDs, respectively. Scale bar, 10 μm. See also Figure S6.
Figure 6
Figure 6. A Subset of TG Synthesis Enzymes Localizes to the Same LDs
(A) GFP-GPAT4 and cherry-AGPAT3 colocalize to the same LD by fluorescence microscopy in cotransfected cells. Scale bars, 10 μm (overview) and 1 μm (magnification). (B) Volcano plot of proteins associating with GPAT4 in the presence or absence of oleate, analyzed by label-free proteomics. The logarithmic ratios of protein intensities are plotted against negative logarithmic p values of the t test performed from three independent experiments. The red dotted line (significance, 0.05) separates specifically interacting proteins marked in black from background (blue dots). All specific interactors are reported in Table S3. (C) Depletion of LD-localized AGPAT4 or DGAT2, but not ER-localized AGPAT1, AGPAT2, or DGAT1, abolishes GPAT4 localization to LDs. Scale bars, 10 μm (overview) and 1 μm (magnification). Western blot analysis shows expression of GPAT4. (D) LDs can acquire GPAT4 after their initial formation, as observed by live-cell 3D time-lapse imaging from 0 to 8 hr of oleate. Scale bars, 1 μm. See also Table S3.
Figure 7
Figure 7. Murine GPAT4 Localizes to LDs and Is Required for the Formation of Large LDs
(A) Murine GPAT4-GFP (green) expressed in COS7 cells relocalizes from the ER to LDs (red) during incubation in oleate-containing medium. Scale bars, 10 μm (overview) and 1 μm (magnification). (B) GPAT4 is required for formation of large LDs specifically under conditions of de novo TG synthesis. Fluorescence images of BODIPY-stained LDs in control of Gpat4−/− BMDMs incubated with oleate or AcLDL-containing medium for 12 hr. Scale bars 10 μm (overview) and 1 μm (magnification). The distribution of diameters for 500 measured LDs from 25 different cells for control (red) or Gpat4−/− (black) is shown. Histogram of LD sizes as in Figure 5A (parameters of Gaussians: a1, 109; b1, 0.4387; c1, 0.1438; a2, 79.14; b2, 0.7446; c2, 0.2606). (C) GPAT4 and DGAT2 are required for large LDs in MEFs. Fluorescence images of BODIPY-stained LDs in control, Gpat4−/−, Dgat2−/−, and Dgat1−/− MEFs incubated with oleate-containing medium for 12 hr. Scale bars, 10 μm (overview) and 2 μm (magnification). The distribution of diameters for 600 measured LDs from 30 different cells for control (red), Gpat4−/− (black), Dgat2−/− (dark green), or Dgat1−/− (light green) is shown (parameters of Gaussians: a1, 85.24; b1, 0.5043; c1, 0.1654; a2, 21.95; b2, 1.067; c2, 0.4563). A TLC showing TG levels in MEFs of the different genotypes is shown. (D) Overexpression of DGAT2 or DGAT1 in MEFs leads to large or small LDs, respectively. Scale bar, 10 μm. (parameters of Gaussians: a1, 161.2; b1, 0.5535; c1, 0.2082; a2, 28.87; b2, 1.654; c2, 1.073). (E) Overexpression of DGAT2 or DGAT1 in murine intestine leads to accumulation of large or small LDs visualized by CARS microscopy, respectively, after an acute dietary lipid challenge (2 hr postgavage with 200 μl oil). Scale bar, 10 μm. See also Movie S2.
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