Figure 1.
Yeast fld1Δ and nine additional mutant strains produce “supersized” LDs (SLDs).
Cells were grown in YPD or SC medium until early stationary phase, stained with Nile red, and examined by fluorescence microscopy. Bar, 5 µm. A). LDs of WT and fld1Δ. B). LDs of mutant strains defective in either CDP-choline pathway (cki1Δ, pct1Δ, and cpt1Δ) or PEMT pathway (cho2Δ, opi3Δ, ino2Δ, and ino4Δ) of PC synthesis. C). Supersized LDs observed in rtc2Δ, mrps35Δ, ckb1Δ and ckb2Δ. D). LDs of a yeast strain with the CDS1 gene under the control of a tetracycline-regulated promoter grown in the presence or absence of doxycycline. E). Transmission electron microscopic examination of LDs in WT, fld1Δ, cho2Δ, opi3Δ, ino2Δ, ino4Δ, and cds1 strains cultured in SC medium. Bar, 1 µm. F). Relative cellular amounts of TAG and SE. #, p<0.05; *, p<0.01, compared to WT.
Table 1.
Yeast gene deletions that lead to the formation of supersized LDs (SLDs).
Figure 2.
Treatment of different phospholipid precursors exerts distinct effects on the formation of SLDs.
WT and mutants were cultured in SC media supplemented without (con) or with 1 mM choline (C), 1 mM ethanolamine (E), or 75 µM inositol (I) to stationary phase and observed under a fluorescence microscope. A). Microscopic images. Bar, 5 µm. B). Percentage of cells containing SLDs. *, p<0.01.
Figure 3.
A link between the formation of SLDs and an elevated level of cellular PA.
A&B). Quantitation of cellular PA in WT and mutants. Cells were grown in SC medium to early stationary phase, harvested, and lyophilized. Lipids were extracted and PA levels were determined by LC-MS. *, p<0.05, compared to WT. C). Overexpression of PAH1 and DPP1 significantly reduces the formation of SLDs in fld1Δ, ino2Δ, and ino4Δ strains. Cells transformed with BG1805-PAH1, BG1805-DPP1 (both from Open Biosystems) or empty vector were cultured in synthetic galactose medium (2% galactose, 0.67% yeast nitrogen base, and amino acids) to stationary phase, stained with Nile red, and examined for the presence of SLDs.
Figure 4.
A) The gene expression fold changes (fld1Δ/WT) for selective genes involved in phospholipid metabolism as determined by microarray analysis. Cells were cultured in YPD medium until log phase (OD600∼0.8). The expression levels of INO1 and OPI3 were significantly upregulated in fld1Δ cells. *, p<0.01 (ANOVA, FDR <0.05). B) Relative mRNA levels of INO1 and OPI3 as determined by qPCR in WT and fld1Δ strains and normalized to ACT1. *, p<0.01. C) WT and fld1Δ cells without or with (+Ino) inositol treatment were grown to late log phase. Microsomes were isolated as described in methods. Lipids were extracted and the amounts of PA were determined by mass-spectrometry. #, p<0.05, compared to WT.
Figure 5.
SLD formation in yeast cells deficient in PA phosphatase activity.
A&B) Formation of SLDs in pah1Δ, but not in dga1Δ or dga1Δ lro1Δ strains. Bar, 5 µm. C) Relative cellular TAG amounts quantified by thin layer chromatography and densitometry. D) Addition of 1 mM membrane-permeable DAG analog (1,2-dioctanoyl-sn-glycerol) and 1 mM oleate, but not oleate alone, significantly elevated the percentage of pah1Δ cells accumulating supersized LDs. Cells with supersized LDs were counted and the percentages were presented as mean±SD.
Figure 6.
PE/PL and PL/TAG ratios of LDs.
A) PE/PL ratio of LDs isolated from WT and mutants grown in SC medium, and of LDs isolated from ino4Δ, fld1Δ, and cds1 cells cultured in SC medium without (con) or with the addition of choline (C), ethanolamine (E), or inositol (I). B) PL/TAG ratio of LDs isolated from WT and mutants grown in SC medium or YPD medium, and of LDs isolated from ino4Δ, fld1Δ, and cds1 cells cultured in SC medium without (con) or with the addition of choline (C), ethanolamine (E), or inositol (I). *, p<0.01; **, p<0.05, compared to WT.
Figure 7.
rtc2Δ and mrps35Δ strains exhibit a higher PE/PC ratio.
WT and mutants were cultured in SC medium to early stationary phase. Cellular amounts of major phospholipid species were analyzed by LC-MS. #, p<0.05; *, p<0.01, compared to WT.
Figure 8.
The mutant strains that produce SLDs display enhanced LD fusion activity in vivo.
Cells were grown in SC media until mid-log phase (OD600∼1.5), stained with Nile red and examined for LD fusion activities under fluorescence microscope. Cells in which two or several LDs lay close together were targeted. Images were taken at a 0.5 s interval. Bar, 5 µm.
Figure 9.
Stability and size of TAG containing artificial droplets consisting of the indicated molar ratios of the phospholipids were determined by light scattering.
The artificial droplets were generated by sonication, and liposomes formed at the same time were removed by density gradient centrifugation. The stability/number of LDs was measured by light scattering. Values are mean ± SD of three experiments.