An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER - PubMed (original) (raw)
An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER
Daniel F Markgraf et al. Cell Rep. 2014.
Abstract
Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Figures
Figure 1. TAG-synthesis and Dga1p-localization on LDs
(A) Pathways connecting neutral lipid and phospholipid metabolism. Enzymes relevant to the present study are highlighted in red. (B) Wild type cells (WT) expressing Dga1p-GFP from a plasmid or Tgl3p-GFP, and _tgl3_Δ mutant cells expressing Dga1p-GFP from a plasmid, were grown to stationary phase and then diluted into fresh medium. At the indicated time points of the growth curve (see Supplemental Figure S1), cells were harvested and analyzed by fluorescence microscopy. Scale bar: 5 μm. (C) TAG levels were determined by TLC and iodine staining in WT (black bars) and _tgl3_Δ (grey bars) cells expressing Dga1p-GFP from a plasmid. Samples were taken at the indicated time points after dilution of stationary cells into fresh medium. Data from three independent experiments are shown as mean +/- SD with the TAG level at t=0 set to 100%. (D) The incorporation of 14C-palmitic acid into TAG was determined. At the indicated time points after dilution of the cells, aliquots were taken and incubated with 14C-palmitic acid for 1h at 30°C.14C-labeled lipids were extracted separated by TLC and quantitated by phosphorimaging and analysis by ImageJ. Labeled TAG was normalized to the total radioactivity in the chloroform-extracted fraction. Data from three independent experiments are shown as mean +/- SD.
Figure 2. Ice2p is a component involved in TAG metabolism
(A) Wild type (WT) or _ice2_Δ mutant cells in stationary phase were diluted into fresh medium lacking or containing 10μg/ml cerulenin, and their growth was followed by measuring the OD at 600nm. Shown are the mean +/- SD of three independent experiments. (B) TAG levels in WT and _ice2_Δ cells, harvested at early exponential phase, were determined after lipid extraction and analysis by mass spectrometry. Data from three independent experiments are shown as mean +/- SD. (C) LDs were stained with BODIPY 493/503 at early exponential phase and analyzed by fluorescence microscopy. Lipid droplets were quantitated using CellProfiler software (n, number of cells analyzed). Scale bar: 5μm.
Figure 3. Ice2p is required for the efficient utilization of LDs during growth resumption
(A) TAG degradation was followed in wild type (WT), _ice2_Δ, and _tgl3_Δ mutant cells after dilution of stationary phase cells into fresh medium containing 10 μg/ml cerulenin. At the indicated time points, lipids were extracted and analyzed by TLC followed by iodine staining. The graphs show quantification of three independent experiments (mean +/- SD) with the TAG level at t=0 set to 100%. (B) As in (A), but stationary cells were diluted into fresh medium without cerulenin. (C) The consumption of LDs in WT and _ice2_Δ cells after dilution from stationary phase into fresh medium containing 10μg/ml cerulenin was followed by BODIPY 493/503 staining and fluorescence microscopy. Scale bar: 10 μm.
Figure 4. Ice2p promotes TAG consumption for phospholipid synthesis
(A)-(E) The lipid composition of wild type (WT) and _ice2_Δ cells was analyzed at different time points after dilution from stationary phase into fresh medium containing 10μg/ml cerulenin. The indicated lipid species were analyzed by quantitative mass spectrometry. Data from two independent experiments were analyzed in duplicates and are shown as mean +/- SD.
Figure 5. Ice2p suppresses a futile cycle between TAG and DAG on LDs
(A) The re-synthesis of TAG in wild type (WT), _ice2_Δ, and _tgl3_Δ mutant cells was analyzed by adding 14C-palmitic acid to cells immediately after their dilution from stationary phase into fresh medium containing 10μg/ml cerulenin. At the indicated time points aliquots were taken, lipids were extracted and analyzed by TLC. Labeled TAG on the TLC plate (left panel) was quantitated by phosphorimaging (right panel). The data were normalized to the total radioactivity in the chloroform-extracted fraction. and are presented as mean +/- SD of four independent expriments. (B) As in (A), but the 14C-palmitic acid was added 2 h after dilution of WT or _ice2_Δ cells. (C) The localization of Dga1p-GFP, expressed from a plasmid, was followed by fluorescence microscopy in WT and _ice2_Δ cells after dilution from stationary phase into fresh medium containing 10μg/ml cerulenin. (D) As in (C), but the localization of Tgl3p-GFP was determined in stationary phase and 4 h after dilution. Scale bars: 5μm.
Figure 6. Growth phase-dependent localization of Ice2p
(A) The localization of Ice2p-GFP was analyzed by fluorescence microscopy at stationary phase and 2h after dilution into fresh medium. (B) The localization of Ice2-GFP was analyzed by fluorescence microscopy in stationary phase cells expressing Erg6-RFP. Six optical sections along the z-axis were collected with a step size of 0.5μm. A maximum projection is shown. Arrows indicate areas of contact between Ice2-GFP and Erg6-RFP labeled LDs. (C) The localization of Ice2p-GFP was analyzed in wild type (WT) and _tgl3_Δ mutant cells in stationary phase and after 1.5 h dilution into fresh medium containing 10μg/ml cerulenin. Scale bars: 5μm.
Figure 7. A cytoplasmic domain of Ice2p binds to lipid droplets
(A) Predicted topology of Ice2p. The largest cytoplasmic loop (amino acids 241-357) is highlighted. (B) Predicted secondary structure of the cytoplasmic loop of Ice2p. Helical wheel representations of predicted helices were generated using the program HeliQuest (
http://heliquest.ipmc.cnrs.fr/
) (C) The localization of a fusion between the cytosolic loop and GFP (Ice2(cyt)-GFP), expressed from a plasmid, was analyzed by fluorescence microscopy in exponentially growing cells. The cells also expressed Erg6p-RFP as a marker for LDs. Scale bar: 5 μm. (D) As in (C), but cells were grown in the presence of 1 mM oleate. (E) The localization of RFP-Ice2(cyt) was analyzed by fluorescence microscopy in COS7 cells. The cells were also stained with BODIPY 493/503 for LDs and for the ER marker calreticulin by immunofluorescence microscopy. Scale bar: 10 μm.
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