Phosphatidylserine decarboxylase

Assembly of lipids into biological membranes is one of the fundamental processes of cell biology to maintain cellular organization and function. During the last few years our laboratory focused on the role of a specific phospholipid, phosphatidylethanolamine (PtdEtn), which turned out to be highly important for various processes including maintenance of membrane integrity. We are using the yeast Saccharomyces cerevisiae as experimental system because this microorganism is well suited for these studies from the view points of molecular biology, biochemistry and cell biology. In the proposed project, we wish to study the enzyme that synthesizes the vast majority of PtdEtn in the yeast, the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p), in some detail. Investigations performed during a previous project (FWF 17321) had demonstrated the central role of Psd1p in yeast lipid metabolism and subcellular distribution of PtdEtn. Surprisingly, the enzyme itself has not been studied at the molecular level. To address this urgent question the following strategy is suggested: (i) First, we wish to extend the enzymatic analysis of Psd1p to a more precise identification and characterization of active and regulatory domains of the enzyme. (ii) Secondly, we will study import and assembly of Psd1p into the inner mitochondrial membrane with emphasis on the role of the subunits and the specific regions (signal and targeting sequences, membrane anchor domains) of this polypeptide. (iii) We will investigate structure and membrane topology of Psd1p specifically addressing the access of the substrate to the active site of the enzyme. A long term and challenging goal will be structural analysis of Psd1p. (iv) Finally, we will study physical, functional and genetic interactions of Psd1p with other mitochondrial/cellular components specifically addressing the role of this enzyme in maintaining mitochondrial and other cellular functions, and in subcellular distribution of PtdEtn and related membrane dynamic processes. These studies are mainly based on promising genetic screenings performed during the previous FWF project 17321. Methods required for these investigations are available in our laboratory or through cooperations with expert partners. We propose that these studies addressing functional and structural properties of Psd1p will shed more light on the molecular role of this enzyme in lipid homeostasis and membrane assembly.

Cellular membranes have gained recently much interest because they harbor a number of important enzymatic processes and at the same time define compartmentation of the cell. Lipids play a major role in membrane biogenesis not only due to their role as physical barrier molecules, but also with respects to their more specific role as modulators of membrane associated biochemical processes. A longstanding interest in our laboratory has been the assembly of lipids into biological membranes using the yeast Saccharomyces cerevisiae as a model cell. Recently, we addressed a specific aspect of this problem which is related to the biosynthesis, subcellular distribution and function of phosphatidylethanolamine (PE). This phospholipid is an important component of cell organelles, especially of mitochondria. Studies of its cell biological properties, however, are challenged by the fact that four different pathways present in three different subcellular compartments contribute to its synthesis. In the FWF project P-21429 we focused on a number of aspects related to Psd1p biochemistry and molecular biology of the yeast. Psd1p, a mitochondrial phosphatidylserine decarboxylase, is the major producer of PE in the yeast. First, we continued our studies of PE transport between yeast organelles with emphasis of the role of production of PE in mitochondria by Psd1p. For this purpose, we investigated PE supply to peroxisomes and studied traffic routes and mechanisms involved. Secondly, we continued our studies of Psd1p interaction with other cellular components on the basis of previous screenings and by interaction of metabolic pathways. Third, we investigated the biogenesis of Psd1p and its assembly into mitochondrial membranes. These studies were meant to shed light on import and assembly of Psd1p into the inner mitochondrial membrane with emphasis on the role of the subunits and specific domains of this polypeptide. These studies also included membrane topology of Psd1p. Finally, we investigated consequences of PE depletion in mitochondria by PSD1 deletion on cellular and mitochondrial functions. These studies were aimed at finding links between functional and structural properties of Psd1p thus affecting lipid homeostasis and membrane assembly.