Sepp Dieter Kohlwein

After three decades, the Yeast Lipid conference returned to Graz, where it all started. This year’s YLC conference centered around the topics: Lipids, Membranes & Organelles; Lipid Droplets: Biogenesis, Storage & Mobilization; Communication via the ‘Lipid Code’; and Yeasts as Cell Factories for Lipids. The organizers, Karin Athenstaedt (University of Graz) and Harald Pichler (Graz University of Technology), were once again able to attract an outstanding panel of plenary speakers, underscoring Graz as a ‘hotspot’ of lipid research and the continued scientific interest in the outlined topics. Short presentations were selected from the submitted abstracts, and almost 40 posters that were on display throughout the conference rounded up an excellent overview of the ‘state-of-the-art’. Poster presenters gave teasers of their work in one-minute flash talks that were a good opportunity to present the essence of their research to all participants, as well as providing a speaking practice. A total number of active attendees of more than 70 (not including one or another retiree with continued fascination in lipids) demonstrated the great international interest in the field, and it was also very rewarding to see so many young yeast researchers, to carry on the enthusiasm about lipid research.

The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21-dependent sensing complex and the activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity, i.e., high-fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.

Lipids are essential biomolecules with diverse biological functions, ranging from building blocks for all biological membranes to energy substrates, signaling molecules, and protein modifiers. Despite advances in lipid analytics by mass spectrometry, the extraction and quantitative analysis of the diverse classes of lipids are still an experimental challenge. Yeast is a model organism that provides several advantages for studying lipid metabolism, because most biosynthetic pathways are well described and a great deal of information is available on the regulatory mechanisms that control lipid homeostasis. In addition, the composition of yeast lipids is much less complex than that of mammalian lipids, making yeast an excellent reference system for studying lipid-associated cell functions.

Thin-layer chromatography (TLC) is a versatile technique for the separation of lipid classes. It provides excellent resolution, can be used for both preparative and analytical applications, and does not require expensive equipment. Here we describe the use of different solvent systems to separate yeast phospholipids and neutral lipids by TLC in one dimension. Resolved lipid species are visualized by iodine vapor or by charring after treatment with sulfuric acid and manganese chloride. Neither of these staining techniques yields a quantitative readout because the mixture of various lipids in yeast affects iodine absorption and charring efficiency; standard curves are required to obtain semiquantitative estimates of the relative lipid composition.

Normal-phase high-performance liquid chromatography (HPLC) is a standard method for separating the major lipid classes in an extract. Owing to the absence of a common property like light absorbance in the various lipid classes, evaporative light-scattering detection (ELSD) is the method of choice for qualitative and quantitative lipid detection. In most cases, neutral lipids and polar lipids are separated by different solvent systems, making it necessary to perform multiple analyses. Compared with other techniques like thin-layer chromatography, normal-phase HPLC–ELSD has better reproducibility and allows a higher degree of automation. Here we describe a method for separating and quantifying yeast neutral lipids and glycerophospholipids in one analytical run.

Analysis of fatty acids by gas chromatography (GC) is facilitated by the generation of volatile fatty acid methyl ester (FAME) derivatives. Here we describe the esterification procedure and a typical program for separating FAMEs using a gas chromatograph equipped with a flame ionization detector or a dual stage quadrupole-mass spectrometry detector. GC is a rather simple technique that provides quantitative information on cellular fatty acid content and composition by use of an internal fatty acid standard.

The diversity of lipid molecules in biological tissues makes their analysis an experimental challenge. Not only do lipids differ greatly in their chemical structures and biophysical properties, but they also occur in greatly varying concentrations in living cells. Accordingly, even for an organism with a relatively simple lipidome such as yeast, multiple extraction and analysis protocols have been developed because none of them allows comprehensive and quantitative determination of all the diverse molecular lipid species. Here we describe an extraction procedure that results in good yields of neutral lipids and glycerophospholipids from yeast. The resulting samples are suitable for analysis by thin-layer chromatography, gas chromatography/mass spectrometry, or high-performance liquid chromatography.