Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

Abstract

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1∆ deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1∆ mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.

Fig. 1

Differential scanning calorimetry of lipid particles. Thermograms showing the second heating scans of LP preparations from wild type W303 (top) and from a hem1Δ mutant (bottom). The thermograms are displaced on the y-axis by arbitrary units for the sake of clarity. The excess heat capacity was normalized to mole SE of the respective samples. Scan rate was 15 °C/h. Analyzed data are listed in Table 2.

Fig. 2

Stress sensitivity of hem1Δ mutants. Cells were grown on YPD plates supplemented with ergosterol and unsaturated fatty acids either at pH 4.0, in the presence of 0.7 M NaCl, or in the presence of 2% dimethylsulfoxide for 72 h. On plates, serial dilutions (steps of 1:10) of cultures are shown starting with a sample of OD₆₀₀ = 1. Growth at pH 8.0 was monitored after 1 week. YPD Erg + UFA, YPD medium with ergosterol and unsaturated fatty acids; DMSO, dimethylsulfoxide.

Fig. 3

Analysis of isolated microsomal membranes. Cells were cultivated to the stationary phase with/without supplements, and microsomes were isolated as described by Zinser and Daum . Proteins were estimated by the method of Lowry et al. , total phospholipids were quantified by the method of Broekhuyse , and sterols were quantified by GLC–MS from aliquots containing a defined amount of protein. Anisotropy was measured as described in Materials and methods. Standard deviation indicated by one asterisk (*) is < 0.0005, and by two asterisks (**) < 0.0025. SQ, squalene; PL, phospholipids; prot, proteins; SFA, saturated fatty acids; UFA, unsaturated fatty acids; ERG, ergosterol; DPH, diphenylhexatriene; r, anisotropy.

Fig. 4

Analysis of isolated plasma membranes. Cells were cultivated to stationary phase with/without supplements, and plasma membrane was isolated as described by Zinser and Daum . For abbreviations see legend to Fig. 3.