Lipid particles of the yeast

Research project P 13669 Lipid particles Günther DAUM 28.06.1999 Lipid particles of the yeast Saccharomyces cerevisiae consist of a core of neutral lipids (triacylglycerols, and steryl esters) which is surrounded by a phospholipid- monolayer with proteins embedded. In the past, these particles were only regarded as a depot of triacylglycerols and steryl esters. During FWF project 11491 we were able to show that lipid particles may have additional functions. Some lipid particle proteins were identified as sterol-synthesizing enzymes, some others as acyltransferases catalyzing formation of phosphatidic acid. These findings and the fact that lipid particles as an organelle have not yet been characterized led us to study this compartment in more detail. Since the protein pattern of lipid particles is relatively simple we succeeded in the identification of the major individual proteins by amino acid sequence analysis. The corresponding genes were determined, and deletion mutants were used to study the phenotypic function and the possible physiological role of the respective gene products. The main subject of the present project is the physiological role and the biogenesis of lipid particles. We will study transport of triacylglycerols and steryl esters from their site of synthesis, most likely the endoplasmic reticulum, to their site of storage. Mechanisms involved in the mobilization of these two lipid species from lipid particle will also be examined. A prerequisite for these studies will be the precise localization of enzymes involved in synthesis and degradation of triacylglycerols and steryl esters. The second major aim of this project will be to study the contribution of lipid particles to cellular lipid synthesis. These investigations will be focused on further identification and characterization of acyltransferases involved in the biosynthesis of phosphatidic acid and characterization of sterol-synthesizing enzymes. Finally, targeting of proteins to the surface of lipid particles will be investigated. Previous experiments had suggested that lipid particles interact with the endoplasmic reticulurn during synthesis and deposition of lipids. This interaction may also be required for protein transport to lipid particles and thus be of general importance for the biogenesis of this compartment. Cell biological and molecular biological methods will be applied to obtain insight into this process at the molecular level.

Lipid particles of the yeast Saccharomyces cerevisiae consist of a core of neutral lipids (triacylglycerols and steryl esters) which is surrounded by a phospholipid monolayer membrane with a small amount and limited number of proteins embedded. Lipid particles were always regarded only as a lipid depot, but during our studies here and in the past the role of this compartment as a lipid biosynthetic organelle was realized. This aspect and the fact that lipid particles as an organelle are poorly studied motivated us to investigate this subcellular fraction in more detail. An important intermediate of lipid biosynthesis is phosphatidic acid. It has been shown before that at least part of the phosphatidic acid biosynthetic pathway is located to lipid particles. Surprisingly, little is know about the formation of this substance at the molecular level, i.e. only some genes of the pathway were identified. Using molecular biological methods we were able to shed some light on this problem during our project. Identification of the 1-acyldihydroxyacetonephosphate acyltransferase Ayr1p was one of the highlights because this was the first enzyme of this kind identified at the molecular level in any type of cell. The observation that phosphatidic acid biosynthesis is distributed among various organelles served as a basis for a new understanding of coordinate and regulated metabolic flux between cellular compartments. Another important lipid class are triacylglycerols. During this project a new gene involved in triacylglycerol synthesis was identified, which was the first enzyme of this kind in the yeast. Similar to enzymes of phosphatidic acid biosynthesis this diacylglycerol acyltransferase is located in part to lipid particles. Triacylglycerols are stored in lipid particles and can be mobilized upon requirement. For this purpose, lipases have to cleave this lipid. Such an enzyme and the corresponding gene were also identified in the course of this project. Lipid particles do not only contain enzymes of phosphatidic acid and triacylglycerol biosynthesis, but also enzymes involved in sterol formation. Some of these enzymes, however, are not only found in one organelle, but are distributed among the lipid particle and the endoplasmic reticulum fraction. In collaboration with two foreign group we studied the interaction of these two compartments during sterol synthesis in yeast. Coordination on the organelle level appears to be of special importance for this pathway because it can be regarded as an efficient means of regulation. Another aspect which evolved from these studies was the targeting of sterol synthesizing enzymes to their "correct" subcellular location. To address this question we employed molecular biological methods which allowed to construct truncated versions of some of these proteins. This strategy was used to search for domains within the proteins which are responsible for the appropriate targeting of the polypeptide to organelles. On the other hand, hybrids of sterol synthesizing proteins with GFP (green fluorescent protein) allowed us to follow the pathway of protein distribution by microscopic methods. These experiments showed that lipid particles interact with the endoplasmic reticulum, the site of the origin of proteins, during their formation. This interaction appears to be the basis for lipid particle biogenesis.