Structural and Biochemical Properties of Lipid Particles from the Yeast Saccharomyces cerevisiae

Tibor Czabany,Andrea Wagner,Dagmar Zweytick,Karl Lohner,Erich Leitner,Elisabeth Ingolic,Günther Daum

doi:10.1074/jbc.m800401200

Abstract

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.

Highlights

Of 95% neutral lipids, TAG, and steryl esters (SE), and only small amounts of phospholipids and proteins [1]
Because wild type and triple mutants (TM) used in this study (Table 1) grow on YPD, TAG and SE were quantified from cells grown to the late logarithmic (16 h) and mid-stationary phase (24 h)
The level of TAG was increased in this strain from the late logarithmic to the mid-stationary phase, it had been argued before that Lrop1p in contrast to Dga1p was more active in logarithmic phase [9]

Summary

EXPERIMENTAL PROCEDURES

Culture Conditions, and Subcellular Fractionation— Yeast strains used throughout this study are listed in the Table 1. When LP fractions were analyzed, samples were delipidated prior to protein quantification For this purpose, nonpolar lipids were extracted with 2 volumes of diethyl ether, and the organic phase was withdrawn; residual diethyl ether was removed under a stream of nitrogen, and proteins were precipitated as described above. The acyl-CoA:diacylglycerol acyltransferase assay was performed in a final volume of 100 ␮l containing 6 nmol of [14C]oleoyl-CoA (80,000 dpm), 0.025 mM dioleoylglycerol, 0.5 mM CHAPS, 150 mM Tris-Cl (pH 7.0), 15 mM KCl, 15 mM MgCl2, and 100 ␮g of yeast cell homogenate [19]. The phospholipid:diacylglycerol acyltransferase assay was performed in the final volume of 100 ␮l containing [14C]palmitic acid-labeled phospholipids (70,000 dpm, 188 nmol), 0.05 mM dioleoylglycerol, 150 mM Tris-Cl (pH 7.0), 15 mM KCl, 15 mM MgCl2, and 200 ␮g of protein [18]. Temperature was controlled with an accuracy of 0.1 °C with

Relevant genotype

RESULTS

Neutral Lipid Product Profile

DISCUSSION