Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy

Abstract

Background: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive.

Results: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response.

Conclusions: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.

Keywords: Autophagy; Glycerolipid; Intracellular trafficking; Organelle; Phospholipid.

Fig. 1

Block in TAG synthesis disrupts the endomembrane system. a Starvation triggers alterations in the ER, Golgi, and mitochondrial morphology in cells defective in TAG synthesis (dga1Δ lro1Δ). Cells expressing indicated organelle markers were transferred from rich medium to nitrogen starvation medium. Organelle morphology was observed by fluorescent microscopy at the indicated time points. Representative images from three independent repeats are shown. DIC, differential interference contrast; Slice, a single slice in the fluorescence z-stack; Projection, max intensity projection of the fluorescence z-stack. Autophagosome*, complete or incomplete autophagosomal structure. Arrows, bulbous structures on the ER. Scale bar, 2 μm. b–d Quantification of organelle defects in a. b Number of ER bulbs per cell. c Percentage of cells displaying abnormal organelle morphology (ER bulb formation, Golgi disappearance, mitochondrial fragmentation). d Progression of autophagy defect as indicated by the decline in the number of GFP-Atg8 dots. Error bar, standard deviation, n = 3. e Inhibition of ER exit leads to disappearance of Golgi and impairment of Atg protein recruitment. sec16-ts cells expressing indicated organelle markers were first grew to mid-log phase under permissive temperature, then transferred to nitrogen starvation medium and incubated under either permissive temperature or non-permissive temperature for 1 h. Images presented as in a. f Quantification of Golgi defects in e. Error bar, standard deviation, n = 3. g Quantification of Atg1 and Atg8 recruitment defects in e. Error bar, standard deviation, n = 3

Fig. 2

Characterization of endomembrane system defects in TAG production defective cells. a The bulbous ER structure contains multiple ER and Golgi proteins. Cells co-expressing two protein chimeras as indicated were starved for 1 h. Representative image slices from individual channels and merged channels are shown. Arrows, bulbous structures on the ER. Scale bar, 2 μm. b The bulbous ER structures are not lipid droplets. Cells expressing Elo3-BFP were starved for 1 h and stained with BODIPY. Images presented as in a. c The ER bulbs are not hollow vesicles. Cells were starved for 1 h. Autophagosomes and ER bulbs were imaged using the Super Resolution via Optical Re-assignment (SORA) technique. Scale bar, 2 μm. d Electron micrograph of yeast cells carrying the indicated genotypes. Cells expressing Emc1-GFP-Apex2 were starved for 1 h and stained with DAB. Representative transmission electron micrographs from two independent repeats are shown. N, nucleus. V, vacuoles. Purple arrows, electron-dense structures connected to the ER. Inserts below, magnified view of demarcated area above and three additional areas from dga1Δ lro1Δ samples, showing the electron-dense structures. Scale bar, 0.5 μm. e Time-lapse imaging of the endomembrane defects. dga1Δ lro1Δ cells were transferred from rich medium to nitrogen starvation medium. Representative image slices (Emc1) or projections (Sec7, Vrg4) at the indicated time point are shown. Time 0 corresponds to 20 min after the medium transfer. Scale bar, 2 μm

Fig. 3

Upregulating phospholipid synthesis or constraining precursor influx rescues endomembrane defects in TAG production defective cells. a, b Phospholipid production was upregulated by (1) knocking out OPI1 or (2) supplying key reactants (inositol, choline, and ethanolamine/ICE). Precursor influx was constrained by (1) 100-fold reduction of glucose supply (0.02% glucose), (2) chemical inhibition of fatty acid synthase (cerulenin), (3) elimination of major fatty acyl-CoA synthetases (faa1Δ faa4Δ), or (4) elimination of a key lysoPA acyltransferase (slc1Δ). a Representative images presented as in Fig. 1a. Scale bar, 2 μm. b Quantification of cells displaying organelle defects. Error bar, standard deviation, n = 3

Fig. 4

Upregulating phospholipid synthesis or constraining precursor influx restores autophagy in TAG production defective cells. Autophagy was assessed by formation of GFP-Atg8 puncta (a, b), formation of Atg1 and Atg5 puncta (c, d), proteolytic processing of GFP-Atg8 (e), and pho8Δ60 assay (f–k). Induction of phospholipid production and reduction of precursor influx were achieved as in Fig. 3. a, c Representative microscopy images presented as in Fig. 1a. Cells starved for 1 h. b, d Quantification of Atg8, Atg1, and Atg5 puncta per cell in a, c. Error bar, standard deviation, n = 3. e Representative immunoblots from three independent repeats. Cells starved for 2 h. f–k pho8Δ60 enzymatic assay. Cells starved for 4 h. Error bar, standard deviation, n = 3

Fig. 5

Accumulation of DAG at the ER in TAG production defective cells. a Starvation triggers intracellular DAG accumulation in dga1Δ lro1Δ cells. Cells of the indicated genotype expressing DAG probes (GFP-PKCδ and GFP-PKCβ) were grown to mid-log phase and then starved for 1 h. Images presented as in Fig. 1a. Yellow arrows, concentration of DAG at the buds in normal cells. Purple arrows, accumulation of DAG at intracellular bulbs. Scale bar, 2 μm. b Starvation triggers DAG accumulation at the ER in dga1Δ lro1Δ cells. Cells treated as in a, except that additional organelle markers (ER, vacuole, late Golgi, and late endosome) were co-expressed. Image presented as in Fig. 2a. Arrows, incidences of GFP-PKCδ colocalization with organelle markers. Scale bar, 2 μm. c, d Total cellular DAG. Cells of the indicated genotype were grown to mid-log phase and then starved for 1 h. Lipids were extracted and analyzed by mass spectrometry-assisted quantification (c) or thin layer chromatography (TLC) (d). c Error bar, standard deviation, n = 3. d Representative image from three independent repeats. e Manipulation of glycerolipid synthesis pathway alters DAG localization. Phospholipid production was upregulated by (1) knocking out OPI1 or (2) supplying key reactants (inositol, choline, and ethanolamine/ICE). Precursor influx was constrained by (1) 100-fold reduction of glucose supply (0.02% glucose), (2) chemical inhibition of fatty acid synthase (cerulenin), or (3) elimination of a key lysoPA acyltransferase (slc1Δ). Cells were starved for 1 h. Images presented as in a. Scale bar, 2 μm. f Exogenous DAG induces endomembrane defects. 1,2-Dioctanoyl-sn-glycerol of the indicated concentrations was added to starvation medium containing 0.02% glucose. Cells were starved for 1 h. Images presented as in Fig. 1a. Scale bar, 2 μm

Fig. 6

Excess DAG, but not PA, is the cause of endomembrane and autophagy defects in TAG production defective cells. a–c Knocking out DGK1 aggravates endomembrane defects in dga1Δ lro1Δ cells. a Representative images presented as in Fig. 1a. Yellow arrows, concentration of DAG at the buds in normal cells. Purple arrows, ER bulbs. Scale bar, 2 μm. b Number of ER bulbs per cell. c Percentage of cells displaying abnormal organelle morphology (ER bulb formation, Golgi disappearance, mitochondrial fragmentation). Error bar, standard deviation, n = 3. d, e Knocking out DGK1 aggravates autophagy defect in dga1Δ lro1Δ cells. Autophagic flux was measured by d proteolytic processing of GFP-Atg8, and e pho8Δ60 assay. Results presented as in Fig. 4e, f. f–h Overexpression of DGK1 and PAH1 produces opposite effects on ER bulb formation in dga1Δ lro1Δ cells. f Representative images presented as in Fig. 1a. Scale bar, 2 μm. g Number of ER bulbs per cell. h Percentage of cells displaying ER bulbs. Error bar, standard deviation, n = 3