Phosphatidylethanolamine, a key lipid of yeast mitochondria

Research project P 14468 Phosphatidylethanolamine Günther DAUM 26.6.2000 The present project is aimed to identify routes of lipid migration into mitochondria and to characterize components involved in this process. The experimental approach is based on the fact that phosphatidylethanolamine (PtdEtn) is an essential component of yeast mitochondrial membranes. PtdEtn can be formed by the mitochondrial Pds1p, the major phosphatidylserine decarboxylase of the yeast. This enzyme relies on the supply with phosphatidylserine (PtdSer) as a substrate from the endoplasmic reticulum. When the mitochondrial PsdIp, is deleted, Psd2p, an extra- mitochondrial isoenzyme of Psd1p, or the extramitochondrial Kennedy pathway (CDP-ethanolamine pathway) are the only sources of mitochondrial PtdEtn. To study the contribution of the two decarboxylases and. the Kennedy pathway to the supply of mitochondria with PtdEtn, mitochond.ria of deletion mutants with defects in the respective pathways will be analyzed for their. lipid patterns. Labeling experiments with radioactive precursors using strains lacking Psd1p, Psd2p or enzymes of the Kennedy pathway will be performed to define and quantify the transport processes involved. Species analysis of mitochondrial PtdSer, PtdEtn and phosphatidy1choline (PtdCho) by chromatographic methods and electrospray tandem mass spectrometry will provide evidence for the specificity of phospholipid species utilized for the formation of mitochondrial membranes. To understand the specific requirement of mitochondria for PtdEtn psdl, psd2 and psdlpsd2 mutant strains will be tested for mitochondrial functions. Membrane properties, correct assembly of membrane components (proteins and lipids), and functional properties of mitochondria will be tested to pinpoint this defect. Lack of mitochondrial PtdEtn formation by deletion of PSDI leads to characteristic phenotypical defects under selective conditions. Similar defects can be expected when the import of PtdSer, the substrate of Psd1p, is impaired. Based on this prediction we propose genetic screenings to detect components governing the import of PtdSer into mitochondria. Different strategies are described for the identification of non-essential, essential and redundant gene products which might govern this process. Yeast is advantageous for this project due to the well- established methods of yeast molecular biology and cell biology.

This project was aimed at studying the cell biological role of phosphatidylethanolamine (PtdEtn), one of the prominent phospholipids from the yeast Saccharomyces cerevisiae, with special emphasis on its function in mitochondria. Molecular biological, cell biological and biochemical methods were employed to address these problems. Our studies unveiled for the first time the essential role of PtdEtn as a cellular and mitochondrial component of the yeast. PtdEtn synthesis in the yeast can be accomplished by three different pathways: (i) decarboxylation of the precursor phosphatidylserine (PtdSer) through catalysis of PtdSer decarboxylase 1 (Psd1p) occurs in mitochondria; (ii) decarboxylation of PtdSer by PtdSer decarboxylase 2 (Psd2p) is located in the Golgi; and (iii) incorporation of ethanolamine via the CDP-ethanolamine branch of the so-called Kennedy pathway is attributed to the endoplasmic reticulum (ER). To estimate the relative contribution of these pathways to the supply of PtdEtn to mitochondrial membranes, labeling experiments in vivo using appropriate precursors and mass spectrometry of products and intermediates were performed with mutants blocked in the respective pathways. These experiments showed that Psd1p is the major source of cellular and mitochondrial PtdEtn, but PtdEtn formed by Psd2p or the CDP-ethanolamine pathway can be imported into mitochondria, although with low efficiency. In contrast to mitochondrial PtdEtn, microsomal PtdEtn is mainly derived from the CDP- ethanolamine pathway. Thus, different pathways of PtdEtn biosynthesis play different roles in the assembly of this phospholipid into cellular membranes. To identify components involved in the maintenance of the mitochondrial PtdEtn level a number of genetic screenings was performed. Different strategies were designed having in mind that proteins facilitating lipid incorporation into mitochondria membranes or proteins regulating this process (i) may be essential, and/or (ii) may occur in redundancy. These screenings relied on the auxotrophy of mutants bearing defects in the different pathways of PtdEtn synthesis, on synthetic lethality of these mutations with defects in additional components involved in homeostasis of PtdEtn in mitochondria, or on the suppression of the defects leading to an improved assembly of PtdEtn into mitochondria. As an alternative, respiratory deficient mutants (petite mutants) were tested for abnormal mitochondrial lipid patterns. All screening strategies described above led to the identification of a number of candidate gene products which are currently subjected to a more detailed functional analysis. Work of this study will be continued in the FWF project 17321.