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. 2019 Sep 23;50(6):755-766.e6.
doi: 10.1016/j.devcel.2019.07.009. Epub 2019 Aug 15.

Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization

Antonio D Barbosa  1 , Koini Lim  2 , Muriel Mari  3 , James R Edgar  1 , Lihi Gal  4 , Peter Sterk  1 , Benjamin J Jenkins  5 , Albert Koulman  5 , David B Savage  2 , Maya Schuldiner  4 , Fulvio Reggiori  3 , Philip A Wigge  6 , Symeon Siniossoglou  7
Affiliations

Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization

Antonio D Barbosa et al. Dev Cell. .

Abstract

Cells dynamically adjust organelle organization in response to growth and environmental cues. This requires regulation of synthesis of phospholipids, the building blocks of organelle membranes, or remodeling of their fatty-acyl (FA) composition. FAs are also the main components of triacyglycerols (TGs), which enable energy storage in lipid droplets. How cells coordinate FA metabolism with organelle biogenesis during cell growth remains unclear. Here, we show that Lro1, an acyltransferase that generates TGs from phospholipid-derived FAs in yeast, relocates from the endoplasmic reticulum to a subdomain of the inner nuclear membrane. Lro1 nuclear targeting is regulated by cell cycle and nutrient starvation signals and is inhibited when the nucleus expands. Lro1 is active at this nuclear subdomain, and its compartmentalization is critical for nuclear integrity. These data suggest that Lro1 nuclear targeting provides a site of TG synthesis, which is coupled with nuclear membrane remodeling.

Keywords: lipid droplet; nuclear membrane; nucleus; phospholipid; triglyceride.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Lro1 Targets a Nuclear Membrane Subdomain that Associates with the Nucleolus (A) Schematic of the major lipid metabolic pathways in yeast; PA, phosphatidate; DG, diacylglycerol; TG, triacylglycerol; FA, fatty acid: LPL, lysophospholipid. (B) Schematic of the PDAT activity; PL, phospholipid. (C) Localization of Lro1-GFP expressed under the control of its own promoter in cells co-expressing an ER (Sec63-mCherry) reporter at the indicated growth phases. (D) Schematic of the organization of the yeast nucleus. (E) Co-localization of Lro1-GFP as in C but with a nucleolar reporter. (F) Left panels: examples of nucleolar enrichment of Lro1-GFP during the exponential phase; right panel, quantification from three experiments, n = 343 cells. (G) Quantification of Lro1 targeting to the nucleolar-associated membrane in response to various stresses. Exponentially growing cells expressing a chromosomally integrated nucleolar reporter (NOP10-mCherry) were subjected to the indicated stresses and the percentage of Lro1-GFP targeting to the nucleolar-associated membrane was quantified (n = 3 experiments, at least 600 cells counted per stress condition); comparisons are between 1 or 2 h and PDS. (H) Immunolabeling of chemically fixed yeast cells expressing Lro1-6xHA. Arrowheads point to gold particles clustering on one side of the nuclear envelope. Stars indicate LDs. MVB, multivesicular bodies; M, mitochondria. Scale bars in (C), (E), and (F), 5 μm; in H, 500 nm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figure S1.
Figure 2
Figure 2
Translocation of Lro1 to the INM that Associates with the Nucleolus (A) Schematic of the topology of Lro1. The K/R-rich nucleolar targeting sequences are shown in red. The Ser324 within the GHSXG lipase motif is shown. (B) lro1Δ cells expressing a nucleolar reporter and the Lro1-GFP mutants shown were imaged at the indicated growth phases. Red stars denote the K/R to A mutations. Arrowheads denote the nucleolus and/or the nucleolar-associated membrane. (C) Quantification of the subcellular localization of the indicated Lro1-GFP mutants in the specified strains. Red stars denote the K/R to A mutations within the extralumenal domain. Three colonies of each strain were analyzed; at least 200 cells were counted for each strain. (D) Lro1-GFP was photobleached, either at the nucleolar-associated membrane or the cortical ER (cER), and fluorescence recovery was measured. Data are means ± SD from three independent experiments (seven cells each); arrow indicates the bleaching event. (E) Nucleolar-associated membrane targeting of 1x-, 2x-, or 3x-MBP-Lro1-GFP fusions during the PDS phase. Right panel: Quantification of the data shown from three experiments, counting only cells with signal in ER or nucleolus; at least 250 cells were quantified for each strain. (F) Localization of the FRB-GFP control (the outlines of cells are shown; vac, vacuole), and the FRB-Lro1-GFP (middle) or FRB-3xMBP-Lro1-GFP (bottom) fusions, before or after the addition of rapamycin. Arrowheads point to the cortical ER membrane. Scale bars in all micrographs, 5 μm. See also Figure S2.
Figure 3
Figure 3
Lro1 Is Catalytically Active at the Nucleolar-Associated INM (A) Wild-type or dga1Δ lro1Δ are1Δ are2Δ (4Δ) cells expressing the indicated plasmids were grown to exponential (EXP) or PDS phases in minimal synthetic medium and spotted on YEPD plates. (B) 4Δ cells expressing Lro1-GFP and Sec63-mCherry were grown from exponential phase to the indicated densities and imaged. (C) 4Δ cells expressing Lro1, or an empty vector, were grown to the indicated densities, labeled with BODIPY 493/503 and their fluorescence was quantified by FACS. Data are representative of two independent experiments. (D) 4Δ cells expressing Lro1-mCherry were grown to the PDS phase and labeled with BODIPY 493/503. Deconvolved through-focus image series were processed to generate 3D image. The full reconstructed field is shown in Video S1. (E) Model for the Lro1-mediated regulation of phospholipid homeostasis; see text for details. (F) Co-localization of the indicated GFP fusions with Lro1-mCherry at the PDS phase. (G) Lipidomic quantifications of TG, LPE, LPC, PE, and PC in wild-type (BY4741), ale1Δ, and plb1Δ plb2Δ plb3Δ nte1Δ lro1Δ (5Δ) cells expressing the denoted plasmids. Cells were grown in galactose for 5 h. Lipid levels were normalized to the corresponding levels of the wild-type strain expressing the empty vector. Data shown are means of at least 5 experiments ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Scale bars in all micrographs, 5 μm. See also Videos S1 and S2.
Figure 4
Figure 4
Effects of Lro1 on Nuclear Morphology (A) Lro1-GFP localization in BY4742 (wild-type), ctf19Δ, and mcm21Δ strains grown to the PDS phase. (B) The BY4741 strain expressing the indicated protein fusions was treated with α-factor; arrowheads point to the nuclear envelope “pocket” that encompasses the nucleolus. (C) BY4741cells expressing PUS1-GFP, NOP1-RFP, and an empty vector or a high-copy GAL-LRO1 plasmid were transferred to galactose-containing medium to induce LRO1 expression, incubated with nocodazole, and extended focal images were collected live. The percentage of arrested cells displaying the elongated nuclear membrane expansion containing the nucleolus (panel 2) was determined; panels 1 shows a typical nucleus without membrane expansion; data shown are means of 5 experiments (at least 360 cells per strain) ± SD. (D) rad52Δ cells expressing the indicated fluorescent fusion proteins and the denoted LRO1 plasmids, were grown at the exponential phase and imaged as above; nuclear circularity of large budded cells was obtained from extended focal images cells as described in STAR Methods; right panels depict circularity measurements from round or expanded rad52Δ nuclei; arrowheads point to the nucleolar-associated membrane expansion; data shown are means of 6 experiments (at least 360 cells per strain) ± SD. (E) A strain carrying an INM anchor (see Figure 2) and expressing PUS1-mCherry and the indicated Lro1 fusions were incubated first with rapamycin, followed by nocodazole. Nuclear circularity was calculated as in D; data shown are means of 3 experiments (at least 260 cells per strain) ± SD. Scale bar for all micrographs, 5 μm. ∗p < 0.05; ∗∗∗p < 0.001. See also Figure S3.
Figure 5
Figure 5
INM Activity of Lro1 Supports TG Synthesis and Is Induced by Availability of Diacylglycerol (A) Schematic of the H1-Lro1 fusion. The Heh1 residues fused to Lro1 are shown; UP, unfolded peptide sequence. (B) Localization of the denoted Lro1-GFP fusions in 4Δ cells. Arrowheads point to the cortical ER membrane. (C) The 4Δ strain expressing either Lro1 or H1-Lro1, or an empty plasmid, was grown to the exponential or PDS phases. Lipids were extracted and TG quantified by mass spectrometry. TG levels shown are relative to internal TG standards of known concentration. Values shown are means from three independent cultures per strain. (D) Upper panel: growth of 4Δ cells in minimal synthetic medium expressing wild-type Lro1, or the indicated Lro1 mutants, in exponential phase or following recovery from the PDS phase. 5-fold dilutions were spotted onto YEPD plates. Lower panel: Survival of 4Δ cells expressing Lro1, or H1-Lro1, in minimal medium. Data are means ± SDs from three different cultures per strain. (E) Exponentially growing 4Δ cells expressing the indicated Lro1 constructs were stained with BODIPY 493/503 to label LDs. (F) 4Δ cells expressing H1-Lro1 and Nup84-mCherry were grown to the PDS phase, stained with BODIPY 493/503, and imaged live using Zeiss LSM880 confocal microscope equipped with an Airyscan unit, as described in STAR Methods, at 0.18 μm axial resolution, and 0.2 μm step slices with 50% overlap. The arrowhead points to a representative intranuclear LD. Arrows point to LDs that associate with the outer nuclear membrane. (G) 4Δ cells expressing H1-Lro1 were grown to the PDS phase and processed for electron microscopy as described in STAR Methods. CW, cell wall; M, mitochondria; N, nucleus; LD is marked with an asterisk. (H) 4Δ cells expressing H1-Lro1 were grown to the PDS phase and processed for high-pressure freezing and freeze substitution as described in STAR Methods. Scale bars in (B) and (E), 5 μm; in (F) and (G), 1 μm; in (H), 500 nm. ∗p < 0.05; ∗∗p < 0.01; ns, not significant. See also Figure S3.
Figure 6
Figure 6
Compartmentalization of INM TG Synthesis Is Required to Maintain Nuclear Homeostasis (A) Distribution of LDs, labeled by BODIPY 493/503, in 4Δ cells expressing Nup84-mCherry and the indicated Lro1 proteins during the PDS phase; the cell outlines are shown. Scale bar, 5 μm. (B) Quantification of the association of LDs with the nuclear envelope in the strains shown in (A) (PDS phase; four experiments, n = at least 350 cells per strain) or in the same strains grown in exponential phase and incubated with glucose-containing media with 0.1% oleate for 2 h (three experiments, n = at least 400 cells per strain); data are means ± SDs. (C) Quantification of nuclear envelope surface area in 4Δ cells expressing the indicated Lro1 proteins at the PDS phase; data are means from five experiments (n = at least 400 per strain counted) ± SDs. (D) Quantification of nuclear envelope circularity in the samples from (A); data are means from six experiments and at least 400 cells per strain. (E) Loss of VPS4 inhibits growth of 4Δ H1-Lro1 cells in the presence of oleate. The indicated strains expressing the Lro1 constructs shown were grown to the exponential phase in glucose-containing medium, spotted on YEPD plates in the absence or presence of 1 mM oleate and grown for 2 days. (F) Loss of CHM7 inhibits growth of 4Δ H1-Lro1 cells in the presence of oleate. The specified strains were grown as described above. (G) Loss of NUP188, but not POM152, inhibits growth of 4Δ H1-Lro1 cells in the presence of oleate. The specified strains were grown as described above. (H) Model for the export of Lro1-derived TG to the outer nuclear membrane; see discussion for details. ∗p < 0.05, ∗∗∗p < 0.001.
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