Karin Athenstaedt

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.

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.

YMR313c/TGL3 Encodes a Novel Triacylglycerol Lipase Located in Lipid Particles of Saccharomyces cerevisiae

Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441–6448) showed that the gene product of YMR313c (named Tgl3p) is a component of yeast lipid particles, and deletion of this gene led to an increase in the cellular level of triacylglycerols (TAG). These observations suggested that TGL3 may encode a TAG lipase of Saccharomyces cerevisiae. Here we demonstrate by cell fractionation and by microscopic inspection of a strain bearing a Tgl3p-GFP hybrid that this polypeptide is highly enriched in the lipid particle fraction but virtually absent from other organelles. The entire TAG lipase activity of lipid particles is attributed to Tgl3p, because the activity in this organelle is completely absent in a Δtgl3 deletion mutant, whereas it is significantly enhanced in a strain overexpressing Tgl3p. A His6-tagged Tgl3p hybrid purified close to homogeneity from a yeast strain overexpressing this fusion protein exhibited high TAG lipase activity. Most importantly, experiments in vivo using the fatty acid synthesis inhibitor cerulenin demonstrated that deletion of TGL3 resulted in a decreased mobilization of TAG from lipid particles. The amino acid sequence deduced from the open reading frame YMR313c contains the consensus sequence motif GXSXG typical for lipolytic enzymes. Otherwise, Tgl3p has no significant sequence homology to other lipases identified so far. In summary, our data identified Tgl3p as a novel yeast TAG lipase at the molecular level and by function in vivo and in vitro.

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.

Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles

Triacylglycerol (TAG) lipases are required for mobilization of TAG stored in lipid particles. Recently, Tgl3p was identified as a major TAG lipase of the yeast Saccharomyces cerevisiae (Athenstaedt, K., and Daum, G. (2003) J. Biol. Chem. 278, 23317–23323). Here, we report the identification of Tgl4p and Tgl5p as additional TAG lipases of the yeast. Both polypeptides, encoded by open reading frames YKR089c/TGL4 and YOR081c/TGL5, share 30 and 26% homology, respectively, to Tgl3p. Cell fractionation experiments and microscopic inspection of strains bearing Tgl4p-GFP and Tgl5p-GFP hybrids demonstrated that both proteins are localized to lipid particles similar to Tgl3p. A 1.7-fold increased amount of TAG enriched in myristic and palmitic acids and the reduced mobilization rate of TAG from tgl4Δ in the presence of the fatty acid synthesis inhibitor cerulenin demonstrated the lipolytic function of Tgl4p in vivo. In contrast, neither the total amount of TAG nor the TAG mobilization rate after addition of cerulenin was affected in tgl5Δ cells. However, the enrichment of C26:0 esterified to TAG of tgl5Δ, an additional increase of TAG in the tgl4Δtgl5Δ double deletion mutant compared with tgl4Δ, and the impairment of TAG mobilization in the tgl4Δtgl5Δ strain in the presence of cerulenin suggested that also Tgl5p functions as a TAG lipase in vivo. Most importantly, the purified His6-tagged Tgl4p and Tgl5p hybrids exhibited TAG lipase activity demonstrating their function in vitro. In summary, our data obtained by biochemical, molecular, and cell biological analyses unambiguously identified Tgl4p and Tgl5p as novel TAG lipases of yeast lipid particles with certain enzymatic specificities.

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.

Loss of Swiss Cheese/Neuropathy Target Esterase Activity Causes Disruption of Phosphatidylcholine Homeostasis and Neuronal and Glial Death in Adult Drosophila

The Drosophila Swiss cheese (sws) mutant is characterized by progressive degeneration of the adult nervous system, glial hyperwrapping, and neuronal apoptosis. The Swiss cheese protein (SWS) shares 39% sequence identity with human neuropathy target esterase (NTE), and a brain-specific deletion of SWS/NTE in mice causes a similar pattern of progressive neuronal degeneration. NTE reacts with organophosphate compounds that cause a paralyzing axonal degeneration in humans and has been shown to degrade endoplasmic reticulum-associated phosphatidylcholine (PtdCho) in cultured mammalian cells. However, its function within the nervous system has remained unknown. Here, we show that both the fly and mouse SWS proteins can rescue the defects that arise in sws mutant flies, whereas a point mutation in the proposed active site cannot restore SWS function. Overexpression of catalytically active SWS caused formation of abnormal intracellular membraneous structures and cell death. Cell-specific expression revealed that not only neurons but also glia depend autonomously on SWS. In wild-type flies, endogenous SWS was detected by immmunohistochemistry in the endoplasmic reticulum (the primary site of PtdCho processing) of neurons and in some glia. sws mutant flies lacked NTE-like esterase activity and had increased levels of PtdCho. Conversely, overexpression of SWS resulted in increased esterase activity and reduced PtdCho. We conclude that SWS is essential for membrane lipid homeostasis and cell survival in both neurons and glia of the adult Drosophila brain and that NTE may play an analogous role in vertebrates.

1-Acyldihydroxyacetone-phosphate Reductase (Ayr1p) of the YeastSaccharomyces cerevisiae Encoded by the Open Reading Frame YIL124w Is a Major Component of Lipid Particles

Biosynthesis of phosphatidic acid through the dihydroxyacetone phosphate pathway requires NADPH-dependent reduction of the intermediate 1-acyldihydroxyacetone phosphate before the second step of acylation. Studies with isolated subcellular fractions of the yeast Saccharomyces cerevisiae revealed that lipid particles and the endoplasmic reticulum harbor 1-acyldihydroxyacetone-phosphate reductase (ADR) activity. Deletion of the open reading frame YIL124w (in the following namedAYR1) abolished reduction of 1-acyldihydroxyacetone phosphate in lipid particles, whereas ADR activity in microsomes of the deletion strain was decreased approximately 3-fold as compared with the wild-type level. This result indicates that (i) both lipid particles and microsomes harbor Ayr1p, which was confirmed by immunological detection of the protein in these two cellular compartments, and (ii) microsomes contain at least one additional ADR activity. As a consequence of this redundancy, deletion of AYR1 neither results in an obvious growth phenotype nor affects the lipid composition of a haploid deletion strain. When a heterozygousAYR1 +/ayr1 − diploid strain was subjected to sporulation; however, spores bearing the ayr1defect failed to germinate, suggesting that Ayr1p plays an essential role at this stage. Overexpression of Ayr1p at a 5- to 10-fold level of wild type caused growth arrest. Heterologous expression of Ayr1p in Escherichia coli resulted in gain of ADR activity in the prokaryote, confirming that YIL124w is the structural gene of the enzyme and does not encode a regulatory or auxiliary component required for reduction of 1-acyldihydroxyacetone phosphate. Taken together, these results identified Ayr1p of the yeast as the first ADR from any organism at the molecular level.

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.

YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylglycerol accumulation. Most interestingly, when oleic acid serves as the carbon source the ratio of triacylglycerol accumulation in mutants to wild-type is significantly increased in strains defective in DGA1 but not in lro1Δ. In vitro experiments revealed that under these conditions an additional acyl-CoA dependent triacylglycerol synthase contributes to triacylglycerol synthesis in the respective mutants. Taken together, evidence is provided that Yarrowia lipolytica contains at least four triacylglycerol synthases, namely Lro1p, Dga1p and two additional triacylglycerol synthases whereof one is acyl-CoA dependent and specifically induced upon growth on oleic acid.