Günther Daum

Intracellular lipid particles of eukaryotic cells

In this review article we describe characterization of intracellular lipid particles of three different eukaryotic species, namely mammalian cells, plants and yeast. Lipid particles of all types of cells share a general structure. A hydrophobic core of neutral lipids is surrounded by a membrane monolayer of phospholipids which contains a minor amount of proteins. Whereas lipid particles from mammalian cells and plants harbor specific classes of polypeptides, mainly perilipins and oleosins, respectively, yeast lipid particles contain a more complex set of enzymes which are involved in lipid biosynthesis. Function of lipid particles as storage compartment and metabolic organelle, and their interaction with other subcellular fractions are discussed. Furthermore, models for the biogenesis of lipid particles are presented and compared among the different species.

Lipids of mitochondria.

A unique organelle for studying membrane biochemistry is the mitochondrion whose functionality depends on a coordinated supply of proteins and lipids. Mitochondria are capable of synthesizing several lipids autonomously such as phosphatidylglycerol, cardiolipin and in part phosphatidylethanolamine, phosphatidic acid and CDP-diacylglycerol. Other mitochondrial membrane lipids such as phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sterols and sphingolipids have to be imported. The mitochondrial lipid composition, the biosynthesis and the import of mitochondrial lipids as well as the regulation of these processes will be main issues of this review article. Furthermore, interactions of lipids and mitochondrial proteins which are highly important for various mitochondrial processes will be discussed. Malfunction or loss of enzymes involved in mitochondrial phospholipid biosynthesis lead to dysfunction of cell respiration, affect the assembly and stability of the mitochondrial protein import machinery and cause abnormal mitochondrial morphology or even lethality. Molecular aspects of these processes as well as diseases related to defects in the formation of mitochondrial membranes will be described.

Synthesis of Triacylglycerols by the Acyl-Coenzyme A:Diacyl-Glycerol Acyltransferase Dga1p in Lipid Particles of the Yeast Saccharomyces cerevisiae

The terminal step of triacylglycerol (TAG) formation in the yeast Saccharomyces cerevisiae is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase (DAGAT). In this study we demonstrate that the gene product of YOR245c, Dga1p, catalyzes a major yeast DAGAT activity which is localized to lipid particles. Enzyme measurements employing a newly established assay containing radioactively labeled diacylglycerol (DAG) as a substrate and unlabeled palmitoyl-CoA as a cosubstrate revealed a 70- to 90-fold enrichment of DAGAT in lipid particles over the homogenate but also a 2- to 3-fold enrichment in endoplasmic reticulum fractions. In a dga1 deletion strain, the DAGAT activity in lipid particles is dramatically reduced, whereas the activity in microsomes is affected only to a minor extent. Thus, we propose the existence of DAGAT isoenzymes in the microsomal fraction. Furthermore, we unveiled an acyl-CoA-independent TAG synthase activity in lipid particles which is distinct from Dga1p and the phosphatidylcholine:DAGAT Lro1p. This acyl-CoA-independent TAG synthase utilizes DAG as an acceptor and free fatty acids as cosubstrates and occurs independently of the acyl-CoA synthases Faa1p to Faa4p. Based on lipid analysis of the respective deletion strains, Lro1p and Dga1p are the major contributors to total cellular TAG synthesis, whereas other TAG synthesizing systems appear to be of minor importance. In conclusion, at least three different pathways are involved in the formation of storage TAG in the yeast.

The life cycle of neutral lipids: synthesis, storage and degradation

Abstract.Triacylglycerols (TAGs), steryl esters (SEs) and wax esters (WEs) form the group of neutral lipids. Whereas TAGs are present in all types of cell, the occurrence of SEs in prokaryotes is questionable, and the presence of WEs as storage molecules is restricted to plants and a few bacteria. Here, we summarize recent knowledge on the formation, storage and degradation of TAGs and SEs in various cell types. We describe the biochemical pathways involved in TAG and SE synthesis and discuss the subcellular compartmentation of these processes. Recently, several novel enzymes governing the metabolism of storage lipids have been identified and characterized. Regulatory aspects of neutral lipid storage are just beginning to be understood. Finally, we describe consequences of defects in neutral lipid metabolism. Since severe diseases like atherosclerosis, obesity and type 2 diabetes are caused by lipid accumulation, mechanisms underlying neutral lipid synthesis, depot formation and mobilization are of major interest for curing such diseases that are increasingly associated with modern civilization.

Characterization of a microsomal subfraction associated with mitochondria of the yeast, Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria

In the yeast, Saccharomyces cerevisiae, similar to higher eukaryotes most phospholipids are synthesized in microsomes. Mitochondria contribute to the cellular biosynthesis of phospholipids insofar as they harbor phosphatidylethanolamine decarboxylase, and enzymes of phosphatidylglycerol and cardiolipin synthesis. In this paper we present evidence that certain enzymes of phospholipid biosynthesis, namely phosphatidylserine and phosphatidylinositol synthase, are enriched in a special microsomal fraction associated with mitochondria, which we named MAM. This fraction was isolated and characterized with respect to marker enzymes, protein and phospholipid composition, and enzymes of phospholipid synthesis. According to these analyses MAMs are a specialized subfraction of the endoplasmic reticulum, which is distinct from other microsomal subfractions. Phosphatidylserine synthesized in MAMs can be readily imported into mitochondria and converted to phosphatidylethanolamine. Reassociation of MAMs with purified mitochondria led to reconstitution of the import of phosphatidylserine into mitochondria. Organelle contact is suggested as a possible mechanism of this process.

Systematic analysis of yeast strains with possible defects in lipid metabolism

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild‐type strains, among them FY1679, CEN.PK2‐1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. Copyright

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

ABSTRACT Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal β-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Δpox2 Δpox3 Δpox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.

YDL142c encodes cardiolipin synthase (Cls1p) and is non-essential for aerobic growth of Saccharomyces cerevisiae

The unassigned open reading frame YDL142c was identified to code for cardiolipin synthase, Cls1p. A cls1 deletion strain is viable on glucose, galactose, ethanol, glycerol and lactate containing media, although the growth rate on non‐fermentable carbon sources is decreased. Mitochondria of the cls1 mutant are devoid of cardiolipin but accumulate the cardiolipin precursor phosphatidylglycerol when grown on non‐fermentable carbon sources. Specific activity of phosphatidylglycerolphosphate synthase in cls1 is reduced to 30–75% of the wild‐type level. Amounts of mitochondrial cytochromes and activity of cytochrome c oxidase, however, are not affected in the cls1 deletion strain. Collectively, these data indicate that cardiolipin is not essential for aerobic growth of Saccharomyces cerevisiae.

Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: lipidome meets proteome.

In the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerols (TG) and steryl esters (SE) are stored in so-called lipid particles/droplets (LP) as biologically inert form of fatty acids and sterols. To understand LP structure and function in more detail we investigated the molecular equipment of this compartment making use of mass spectrometric analysis of lipids (TG, SE, phospholipids) and proteins. We addressed the question whether or not lipid and protein composition of LP influence each other and performed analyses of LP from cells grown on two different carbon sources, glucose and oleate. Growth of cells on oleate caused dramatic cellular changes including accumulation of TG at the expense of SE, enhanced the amount of glycerophospholipids and strongly increased the degree of unsaturation in all lipid classes. Most interestingly, oleate as a carbon source led to adaptation of the LP proteome resulting in the appearance of several novel LP proteins. Localization of these new LP proteins was confirmed by cell fractionation. Proteomes of LP variants from cells grown on glucose or oleate, respectively, were compared and are discussed with emphasis on the different groups of proteins detected through this analysis. In summary, we demonstrate flexibility of the yeast LP lipidome and proteome and the ability of LP to adapt to environmental changes.

The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing

The novel Drosophila mutant löchrig (loe) shows progressive neurodegeneration and neuronal cell death, in addition to a low level of cholesterol ester. loe affects a specific isoform of the γ‐subunit of AMP‐activated protein kinase (AMPK), a negative regulator of hydroxymethylglutaryl (HMG)‐CoA reductase and chol esterol synthesis in vertebrates. Although Drosophila cannot synthesize cholesterol de novo, the regulatory role of fly AMPK on HMG‐CoA reductase is conserved. The loe phenotype is modified by the level of HMG‐CoA reductase and suppressed by the inhibition of this enzyme by statin, which has been used for the treatment of Alzheimer patients. In addition, the degenerative phenotype of loe is enhanced by a mutation in amyloid precursor protein‐like (APPL), the fly homolog of the human amyloid precursor protein involved in Alzheimers disease. Western analysis revealed that the loe mutation reduces APPL processing, whereas overexpression of Loe increases it. These results describe a novel function of AMPK in neurodegeneration and APPL/APP processing which could be mediated through HMG‐CoA reductase and cholesterol ester.