Systematic analysis of the role of phospholipid metabolism in nuclear/ER membrane structure. A, pathways of phospholipid biosynthesis and PA metabolism in budding yeast. Reactions involved in the production of cellular pools of phospholipids are shown. For simplicity, the Kennedy pathway reactions leading to PE synthesis are not shown. Blue and red arrows highlight the CDP-DAG and CDP-choline (Cho) pathways, respectively. G3P, glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate. LPA, lysophosphatidic acid; PI, phosphatidylinositol; PS, phosphatidylserine. B, list of mutants tested for nuclear/ER morphology defects. Cells were transformed with a centromeric plasmid expressing SEC63-GFP and visualized by microscopy. At least two transformants per mutant were analyzed. +, no defects in nuclear or ER structure; -, severe defects; +/-, partial defects. The tet-CDS1 repressible strain was grown in the presence of 10 μg/ml doxycycline, and SEC63-GFP fluorescence was visualized after 0, 6, 12, and 24 h. The pis1 temperature-sensitive strain was grown at 30 °C and then transferred to 37 °C for 3 h. All mutants were derived from BY4742, except the tet-CDS1 and psd1Δ strains, which were derived from BY4741, and the pis1 (41) and pah1Δ (10) strains. C, representative images of wild-type, tet-CDS1 (12 h in doxycycline), and opi3Δ expressing the SEC63-GFP reporter. Scale bar = 5 μm.

DGK1 rescues the lethality of dephosphorylated PAH1. A, identification of DGK1 as a high copy suppressor of the inositol auxotrophy of PAH1-7P. The wild-type RS453 strain carrying a high copy vector expressing the non-phosphorylated PAH1-7P mutant from a galactose-inducible promoter was transformed with the indicated plasmids. Transformants were grown on synthetic plates lacking inositol and supplemented with glucose (left panel) or galactose (right panel) at 30 °C. B, identification of DGK1 as a high copy suppressor of the lethality of NEM1-SPO7 overexpression. RS453 cells carrying plasmids coexpressing NEM1 and SPO7 from a galactose-inducible promoter were transformed with the indicated plasmids. Transformants were grown on synthetic plates supplemented with glucose (left panel) or galactose (right panel) at 30°C. C, primary structure and hydropathy plot of Dgk1p. The gray box indicates the conserved cytidylyltransferase domain within Dgk1p. Below, the hydropathy plot of Dgk1p is given (42), using a window of 19 amino acids. The black lines indicate the positions of the predicted transmembrane domains. D, GFP-Dgk1p localizes to the nuclear membrane and ER. A GFP-DGK1 fusion under the control of the DGK1 promoter was expressed from a centromeric plasmid in a dgk1Δ strain. Cells were grown in early logarithmic phase and visualized by fluorescent microscopy. Scale bar =5 μm.

DGK1 opposes the function of PAH1 at the nuclear membrane. A, overexpression of DGK1 causes nuclear/ER membrane proliferation and nuclear expansion. The RS453 strain expressing the plasmid-borne SEC63-GFP and RFP-PUS1 reporters was transformed with a centromeric plasmid expressing DGK1 under the control of a galactose-inducible promoter or an empty vector. Cells were grown in galactose-containing medium and visualized by confocal microscopy. Scale bar = 5 μm. B, deletion of DGK1 restores normal nuclear structure in pah1Δ cells. Wild-type RS453, pah1Δ, and pah1Δ dgk1Δ cells transformed with a centromeric plasmid expressing SEC63-GFP were visualized by confocal microscopy. Scale bar = 5 μm.

Deletion of DGK1 restores normal PA and INO1 transcript levels in pah1Δ cells. A, phospholipid composition of wild-type, dgk1Δ, pah1Δ, and pah1Δ dgk1Δ cells in the exponential phase of growth. The indicated yeast strains were grown to exponential phase in the presence of ³²P_i (10 μCi/ml). Phospholipids were extracted and separated by two-dimensional thin layer chromatography. Images of ³²P-labeled phospholipids were visualized by phosphorimaging and subjected to ImageQuant analysis. The percentages shown for the individual phospholipids were normalized to the total ³²P-labeled chloroform-soluble fraction that included sphingolipids and unidentified phospholipids. Each data point represents the mean ± S.D. of three experiments. PI, phosphatidylinositol; PS, phosphatidylserine. B, neutral lipid composition of wild-type, dgk1Δ, pah1Δ, and pah1Δ dgk1Δ cells in the stationary phase of growth. The indicated yeast strains were grown to stationary phase in the presence of [2-¹⁴C]acetate (1 μCi/ml). Lipids were extracted and separated by one-dimensional thin layer chromatography. Images of ¹⁴C-labeled lipids were visualized by phosphorimaging and subjected to ImageQuant analysis. The percentages shown for the individual lipids were normalized to the total ¹⁴C-labeled chloroform-soluble fraction. Each data point represents the mean ± S.D. of three experiments. TAG, triacylglycerol; Erg, ergosterol; ErgE, ergosterol ester; FA, fatty acid; PL, phospholipid. C, INO1 mRNA levels of wild-type, pah1Δ, dgk1Δ, and pah1Δ dgk1Δ cells. The mRNA levels of INO1 were analyzed in the indicated strains grown in YEPD medium by quantitative real-time reverse transcription-PCR. Amplification of each sample was performed in duplicate and normalized to RTG2. Values and errors were calculated from three independent experiments. Values are expressed as -fold derepression over the values measured in the wild-type strain.

Dgk1p is a novel CTP-dependent DAG kinase. A, the dgk1Δ mutation abolishes diacylglycerol kinase activity. Cell extracts were prepared from wild-type, dgk1Δ, pah1Δ, and pah1Δ dgk1Δ cells at the exponential phase of growth, and 50 μg of protein from each cell extract was assayed for diacylglycerol kinase activity. Left panel, the ³²P-labeled reaction product was extracted with chloroform, separated by thin layer chromatography using a solvent system of chloroform/methanol/water (65:25:4), and visualized by phosphorimaging. Right panel, the ³²P-labeled reaction product was extracted with chloroform and subjected to liquid scintillation counting. Each data point represents the mean ± S.D. of triplicate enzyme determinations. B, Dgk1p exhibits DAG kinase activity. The DAG kinase activity of partially purified Dgk1p was measured with the indicated protein content (left panel) and for the indicated time intervals (right panel).

The DAG kinase activity of Dgk1p is required for nuclear membrane expansion. A, multiple sequence alignment of Dgk1p (residues 162-182) with cytidylyltransferases from the indicated species. The first residue of each sequence segment is indicated. The arrow points to the conserved aspartate residue. B, the DGK1(D177A) mutant lacks DAG kinase activity. The dgk1Δ strain was transformed with an empty centromeric plasmid or with the same plasmid carrying DGK1 or DGK1(D177A) under the control of the GAL1/10 promoter. Cell extracts from the corresponding strains were assayed for diacylglycerol kinase activity. Values and errors were calculated from three experiments. C, DGK1(D177A) does not promote nuclear membrane growth. The RS453 strain expressing a SEC63-GFP fusion was transformed with the galactose-inducible DGK1 or DGK1(D177A) construct from B, grown to early logarithmic phase in galactose-containing medium, and inspected by microscopy.

Overexpression of Dgk1p causes relocation of a PA-binding reporter at the nuclear membrane. The RS453 strain expressing the mCherry-PUS1 fusion and the GFP-SPO20-(51-91) reporter was transformed with a 2 μ vector (A) or the same vector carrying DGK1 (B). Cells were grown in raffinose, transferred into galactose-containing medium for 6 h, and viewed by confocal microscopy. Scale bar = 5 μm.

Overexpression of Dgk1p causes proliferation of the nuclear membrane but not cortical ER membrane. A, shown is the time course of the induction of DGK1-PtA. Wild-type cells (RS453) expressing DGK1-PtA under the control of the GAL1/10 promoter in a centromeric plasmid were transferred from raffinose to galactose-containing medium, and protein extracts were prepared at the indicated time points. Protein samples were resolved by SDS-PAGE followed by Western blotting using anti-PtA antibodies. B, cells expressing an HEH2-GFP fusion and DGK1 under the control of the GAL1/10 promoter in a centromeric plasmid were transferred from raffinose to galactose-containing medium for the indicated times. Heh2p-GFP labeling (upper panels) or cell/Heh2p-GFP overlays (lower panels) were visualized at the indicated time points by confocal microscopy. C, cells expressing an RTN2-GFP fusion and DGK1 under the control of the GAL1/10 promoter in a centromeric plasmid were transferred from raffinose to galactose-containing medium for the indicated times. Upper panels, confocal Z-sections corresponding to the cell center; lower panels, confocal sections corresponding to the cell periphery. Scale bars = 5 μm.

Genetic interactions of phospholipid biosynthetic mutants with pah1Δ indicate a role for PA in nuclear membrane proliferation. A, deletion of GAT2 partially rescues the growth defects of pah1Δ. Serial dilutions of the indicated strains were spotted on YEPD plates and grown for 2 days at 30 and 37 °C. B, deletion of GAT2 decreases INO1 mRNA levels in the pah1Δ cells. The mRNA levels of INO1 were analyzed in the indicated strains grown in YEPD medium by quantitative real-time PCR. Amplification of each sample was performed in duplicate and normalized to RTG2. Values and errors were calculated from three independent experiments. The values indicated within the bars represent the -fold derepression over the values measured in the wild-type strain. C, the GAT2-dependent rescue depends on its catalytic activity. Serial dilutions of the indicated strains were spotted on synthetic plates and grown for 2 days at 30 and 37 °C. D, lack of active GAT2 partially rescues nuclear membrane proliferation in pah1Δ cells. The pah1Δ gat2Δ mutant expressing SEC63-GFP was transformed with the indicated plasmids, and cells were observed by confocal microscopy. Scale bar = 5 μm. E, deletion of PSD1, CHO2, and OPI3 does not rescue the growth defects of pah1Δ. Serial dilutions of the indicated strains were spotted on -URA (left panel) or 5-fluoroorotic acid (5-FOA; right panel) plates and grown for 2 or 3 days, respectively, at 30 °C.