Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae

Abstract

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinones. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Delta mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Delta deletion in a dga1Delta lro1Delta are1Delta are2Delta quadruple mutant background (QMhem1Delta), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Delta mutant in a wild type background. In QMhem1Delta, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Delta did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes.

FIGURE 1.

Heme dependence of ergosterol synthesis. The yeast sterol-14-α-demethylase (Erg11p) is a cytochrome P450 protein and depends on heme. Deletion of HEM1 can be overcome by supplementing yeast cells with δALA.

FIGURE 2.

Sterol composition and squalene accumulation in hem1Δ mutants. A, wild type BY4742 (gray bar), hem1Δ + δALA (white bar), and hem1Δ + ergosterol + Tween 80 (black bar). B, QM dga1Δlro1Δare1Δare2Δ (gray bar), QMhem1Δ + δALA (white bar), and QMhem1Δ + ergosterol + Tween 80 (black bar). Cells were grown to the stationary phase, lipids were extracted, and sterols/squalene were quantified by GLC-MS. Data are mean values of five independent experiments. Error bars, S.D. values. *, in these samples, only marginal amounts of the respective compound were detected. SQ, squalene; ERG, ergosterol; LAN, lanosterol.

FIGURE 3.

Visualization of lipid storage compartments by fluorescence microscopy. Wild type BY4742 (A), hem1Δ (B), QM (C), and QMhem1Δ (D) were grown to the stationary phase, stained with Nile Red, and inspected by fluorescent microscope as described under “Experimental Procedures.” Bar, 10 μm.

FIGURE 4.

Overproduction of squalene in a quadruple mutant dga1Δlro1Δare1Δare2Δ does not induce lipid particle formation. Wild type (A), hem1Δ (B), QM (C), and QMhem1Δ (D) were grown to the stationary phase and then processed and inspected by electron microscopy as described under “Experimental Procedures.” Bar, 1 μm. LP, lipid particle.