Staff Members of the Institute of Biochemistry, TU - Institut für ...

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Previous investigations in our laboratory were aimed at the molecular biological identification of novel components involved in PE homeostasis of the yeast Saccharomyces cerevisiae. For this purpose, a number of genetic screenings were performed. To obtain a global view of the role of PE in the cell and to study the effects of an unbalanced PE level we recently subjected a psd1∆ deletion mutant and the corresponding wild type to DNA microarray analysis and examined genome-wide changes in gene expression caused by a PSD1 deletion. Comparison of the gene expression pattern of the psd1∆ mutant with the wild-type led to the identification of 55 differentially expressed genes. Grouping of these genes into functional categories revealed that PE formation by Psd1p influenced the expression of genes involved in diverse cellular pathways including transport, carbohydrate metabolism and stress response. Most of the proteins identified are present in the cytosol followed by the nucleus, cellular membranes, the cell wall and mitochondria. Moreover, this genome-wide analysis identified a number of gene products with unknown function which are currently subjected to detailed molecular analysis addressing PE homeostasis in the yeast. 16 Figure 1: Three-dimensional reconstruction of yeast cells grown on oleic acid. Transmission electron micrographs of ultrathin sections (A, C; bar = 1µm) and corresponding 3D reconstructions of serial sections (B, D) of a chemically fixed wild type cell are shown. Cells shown in A and B were grown on YPD, and cells shown in C and D were grown on oleic acid to induce formation of peroxisomes. CW cell wall; ER endoplasmic reticulum; LP lipid particle; M mitochondrion; N nucleus; V vacuole; PX peroxisomes. By courtesy of G. Zellnig, KFUG. To understand cellular PE homeostasis in some more detail, we performed experiments defining traffic routes of PE within the yeast cell. In recent studies, we investigated peroxisomes and the plasma membrane as destinations for PE distribution. These two membranes have in common their lack of capacity to synthesize PE. We employed yeast mutants bearing defects in the different pathways of PE synthesis and demonstrated that PE formed though all four pathways can be supplied to peroxisomes and to the plasma membrane. The fatty acid composition of peroxisomal and plasma membrane phospholipids was mostly influenced by the available pool of total species synthesized, although a certain balancing effect was observed regarding the assembly of PE species into the two target compartments. We assume that the phospholipid composition of peroxisomes and the plasma membrane is mainly affected by the synthesis of the respective components and subject to equilibrium, and to a lesser extent affected by specific transport and assembly processes.
Organelle association and membrane contact between organelles (Fig. 1) may be a relevant mechanism for lipid transport between organelles, but components governing this process and related events have to be studied in future investigations. Growth of yeast on oleic acid not only induces formation of peroxisomes, but also leads to an intense synthesis of triacylglycerols which are stored in lipid particles (see below). The link between neutral lipid metabolism and peroxisome proliferation is subject to current investigations with emphasis on the role of enzymes involved. These studied are related to the possible lipotoxic effect of fatty acids in yeast. Most recently we also studied the link between PE metabolism and neutral lipid storage. For this purpose we analyzed lipids from strains bearing defects in PE synthesis. These analyses showed that a mutant bearing a defect in the CDP-ethanolamine pathway had a decreased level of triacylglycerols (TAG). The molecular reason for this finding is currently under investigation. Neutral lipid storage in lipid particles and mobilization Yeast cells have the capacity to store neutral lipids TAG and STE (steryl esters) in subcellular structures named lipid particles/droplets. Upon requirement, TAG and STE can be mobilized and serve as building blocks for membrane biosynthesis. In an ongoing project of our laboratory, we focused on the characterization of enzymatic steps which lead to the mobilization of TAG and STE depots. In Saccharomyces cerevisiae, the three STE hydrolases Tgl1p, Yeh1p and Yeh2p contribute differently to STE mobilization from their site of storage, the lipid particles. In a biochemical study we investigated enzymatic and cellular properties of these three hydrolytic enzymes. Using the respective single, double and triple deletion mutants and strains overexpressing the three enzymes, we demonstrated that each STE hydrolase exhibits certain substrate specificity. Interestingly, disturbance in STE mobilization also affects sterol biosynthesis in a type of feedback regulation. We also showed that sterol intermediates stored in STE and set free by STE hydrolases are recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol. This recycling implies that the vast majority of sterol precursors are transported from lipid particles to the ER where sterol biosynthesis is completed. Ergosterol formed through this route is then supplied to its subcellular destinations, especially the plasma membrane. Only a minor amount of sterol precursors set free from STE are randomly distributed within the cell after cleavage. In conclusion, STE storage and mobilization although dispensable for yeast viability contribute markedly to sterol homeostasis and distribution. A major focus of our neutral lipid project was the biochemical characterization of the three yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast TAG lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out biochemical experiments with wild type and deletion strains bearing defects in Tgl3p, Tgl4p and Tgl5p. We demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected 17
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