Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast - PubMed (original) (raw)
Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast
Julia Petschnigg et al. J Biol Chem. 2009.
Abstract
Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.
Figures
FIGURE 1.
_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutant lacks lipid droplets and exhibits growth defects. A, Nile Red staining of wild-type and the _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ mutant YJP1078, discrimination of LD and membranous structures based on different fluorescence emission characteristics of Nile Red. Left panels, λex/λem excitation/emission at 543/550–570 nm detects preferentially LD; these are completely absent in the mutant (lower row). Right panels, λex/λem 543/600–650 nm fluorescence emission range detects, in addition to LD in wild type, labeled membranous structures both in wild type and in the mutant. Scale bar, 5 μm. B, growth (cell number/ml) of wild type, _dga1_Δ _lro1_Δ double and _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutants in complete media. ♦, wild type; ■, _dga1_Δ lro1; ▲, _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ. Growth of _dga1_Δ _lro1_Δ and the quadruple mutants is severely delayed during the first 7 h of cultivation (inset). All cell types reach similar cell densities in stationary phase.
FIGURE 2.
_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutant is highly sensitive to exogenously supplied oleic and palmitoleic acid. A, growth tests of yeast strains on agar plates containing different FA. Wild-type, _are1_Δ _are2_Δ, _dga1_Δ _lro1_Δ, _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ, _DGA1_[_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ] (quadruple mutant transformed with a plasmid expressing DGA1 wild-type gene), and _LRO1_[_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ] (quadruple mutant transformed with a plasmid expressing the LRO1 gene) were grown to stationary phase and diluted to _A_600 = 0.5, and serial dilutions of 1:10 were spotted onto agar plates containing different FA dissolved in 1% Brij58. Palmitic acid (C16:0), stearic acid (C18:0), palmitoleic acid (C16:1), and oleic acid (_C18:_1), control is in the presence of 1% Brij58 only. w/o, without FA supplementation. B, determination of cell vitality of yeast strains on FA addition using SytoxGreenTM staining. Wild-type (top panel), _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ (1st middle panel), and _DGA1_[_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ] and _LRO1_[_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ] (2nd middle panel) were grown overnight, shifted to fresh YND medium, and grown for 4 h. Cells were incubated in the presence of indicated FA (0.001%) for 12 h prior to SytoxGreenTM staining. FA toxicity is most pronounced for oleate > palmitoleate and not detectable on supplementation with saturated FA. Dga1p and, to a lesser extent, Lro1p expression in the quadruple mutant rescues unsaturated FA toxicity. Addition of palmitic acid (bottom panel) restored viability to the quadruple mutant, in the presence of oleic acid. Scale bar, 10 μm; DIC, differential interference contrast.
FIGURE 3.
_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutant displays massive membrane proliferations on unsaturated fatty acid treatment. A, transmission image (differential interference contrast (DIC)) of _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutants treated with 0.001% oleic acid for 3 h. Note the appearance of highly light diffractive structures (arrowheads) corresponding to membrane proliferations. B, visualization of membrane proliferations in the _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ mutant treated with fatty acids. The quadruple mutant was grown for 4 h prior to treatment (3 h) with palmitic, oleic, and palmitoleic acid (0.001% each); wild-type treated with 0.001% oleic acid for 3 h served as a control. Upper row, Nile Red staining in the mutant shows the appearance of massive membrane proliferations detectable in the 600–650 nm emission range, on unsaturated FA supplementation. Lower row, expression of Elo3-GFP confirms the rearrangement and proliferation of the ER membrane in the mutant exposed to unsaturated FA. Scale bar, 5 μm. C, electron micrographs of wild-type and mutant cells. Cells were grown in minimal medium in the absence or presence of 0.001% FA. Lipid droplets are readily detectable in the wild type grown in the presence of oleic acid but are absent from the _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutant under all conditions analyzed. Addition of oleic acid induced massive membrane proliferations in the quadruple mutant (arrowheads). M, mitochondrion; N, nucleus; ER, endoplasmic reticulum; LD, lipid droplet; V, vacuole; w/o, without FA supplementation. Scale bar, 1 μm.
FIGURE 4.
Proliferation of membranes in the _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutant is a fast response to oleic acid addition and independent of the unfolded protein response and does not directly correlate with a loss of viability. A, quadruple mutant cells and _DGA1_[_are1_Δ _are2_Δ _dga1_Δ _lro1_Δ] cells were grown overnight, transferred to fresh medium for 4 h, and treated with 0.001% oleic acid for the indicated time points. Staining with Nile Red and detection in the 600–650 nm fluorescence emission range demonstrate that appearance of membrane proliferations precedes loss of viability (indicated by SytoxGreenTM staining) by about 2 h. Scale bar, 5 μm. DIC, differential interference contrast. B, wild-type and mutant cells harboring a UPRE-lacZ construct were cultivated overnight and grown for 4 h in fresh SD media prior to 0.001% oleic acid addition. At indicated time points, aliquots were withdrawn and subjected to β-galactosidase assay. Activity (fold increase; black bars in the presence of oleate) is given relative to untreated wild-type cells (white bars) at time point 0. Assays were performed in triplicate. C, growth tests of yeast strains on agar plates containing different FA. Wild-type, _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ quadruple mutants, and _ire1_Δ _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ pentuple mutants were grown to stationary phase and diluted to _A_600 = 0.5, and serial dilutions of 1:10 were spotted onto agar plates containing oleic acid dissolved in 1% Brij58 at indicated concentrations. Additional deletion of the IRE1 gene in the quadruple mutant background does not affect fatty acid sensitivity of the strain. D, membrane proliferations also occur in the _ire1_Δ _are1_Δ _are2_Δ _dga1_Δ _lro1_Δ pentuple mutant treated with oleic acid. The pentuple mutant was grown for 4 h prior to treatment with 0.001% oleic acid. Nile Red staining and fluorescence detection in the 600–650 nm range shows massive membrane proliferations in the mutant on oleic acid supplementation, within 30–60 min. Scale bar, 5 μm. E, electron micrographs of the pentuple mutant treated with oleic acid. Cells were grown in minimal medium for 4 h prior to oleic acid addition (0.001%) for 3 h. Induction of membrane proliferations (arrowheads) in the _ire1_Δ _are1_Δ _are2_Δ _dga1_Δ lro1_Δ pentuple mutant on oleic acid is very rapid and independent of Ire1p. Note the altered morphology of these membrane proliferations, compared with the quadruple mutant (Fig. 3_C), which show striking similarity to ERAC structures (73). V, vacuole; ER, endoplasmic reticulum; N, nucleus; w/o, without FA supplementation. Scale bar, 1 μm.
FIGURE 5.
ER-to-Golgi trafficking is blocked in the quadruple mutant on addition of oleic acid. Cells were grown overnight, shifted to fresh medium, and incubated for 4 h. FA were added at concentrations of 0.001%, and cells were harvested at indicated time points. Proteins were extracted and separated by 8% SDS-PAGE. Carboxypeptidase Y processing was monitored using a CPY-specific antibody (Rockland Inc.). proCPY, 67-kDa ER form; mCPY, mature 61-kDa vacuolar form; w/o, without FA supplementation. Anti-Tcm (ribosomal protein) was used as a loading control.
References
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