Phosphatidylserine decarboxylase 1 of the yeast (continued)

The mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) of the yeast is a central and important enzyme in cellular lipid metabolism. Psd1p which consists of an a- and a ß-subunit catalyzes formation of phosphatidylethanolamine (PE) by decarboxylation of phosphatidylserine (PS). During the last few years work in our lab was focused on (i) the role of Psd1p in the lipid biosynthetic network; (ii) biosynthesis, processing and assembly of Psd1p into mitochondrial membranes; and (iii) the role of PE as an essential phospholipid for the maintenance of membrane integrity, especially of mitochondria. In the project proposed here we will continue and extend these investigations to obtain a deeper insight into molecular properties of Psd1p. For this purpose, we will dissect functions and features of specific domains of Psd1p such as membrane anchors, membrane targeting domains, substrate recognition sites, the active center at the a/ß-subunit contact zone and further sequence stretches potentially involved in correct processing and membrane assembly of the enzyme. Moreover, we will investigate the structure and topology of Psd1p with emphasis on membrane assembly, protein complex formation in the inner mitochondrial membrane and access of the substrate to the enzyme. Methods required for these investigations are available in our laboratory or through the expertise of our cooperation partners. These studies addressing the link between functional and structural properties of Psd1p will contribute substantially to our understanding of the molecular role of Psd1p in lipid homeostasis and membrane biogenesis.

Phosphatidylethanolamine (PE) is one of the major phospholipids of yeast membranes as it is highly important for membrane stability and integrity. PE synthesis in the yeast is accomplished by four different pathways, namely (i) synthesis of phosphatidylserine (PS) in the endoplasmic reticulum and decarboxylation by the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p); (ii) synthesis of PS and conversion to PE by the Golgi localized Psd2p; (iii) the CDP-ethanolamine pathway (Kennedy pathway) in the endoplasmic reticulum, and (iv) the lysophospholipid acylation route catalyzed by Ale1p and Tgl3p. The major player in the yeast PE biosynthetic network is phosphatidylserine decarboxylase 1 (Psd1p) of the inner mitochondrial membrane (IMM), an enzyme forming PE by decarboxylation of phosphatidylserine (PS). A number of cellular functions, especially in mitochondria, require the activity of Psd1p. A specific feature of Psd1p is its structural arrangement in the form of an ?- and a ?-subunit. The latter domain anchors the protein to the inner mitochondrial membrane, whereas the ?-subunit which is separated from the ?-subunit during membrane assembly and largely responsible for the enzymatic activity is per se soluble. The ? -subunit harbors a highly conserved motif, which was proposed to be involved in phosphatidylserine (PS) binding. In a recent study we performed a molecular analysis of this consensus motif for the function of Psd1p by using Psd1p variants bearing either deletions or point mutations in this region. Our data show that mutations in this motif affect processing and stability of Psd1p, and consequently the enzymes activity. Thus, we conclude that this consensus motif is essential for structural integrity and processing of Psd1p.We also investigated the membrane anchoring of Psd1p in the inner mitochondrial membrane. We found that Psd1p harbors at least two membrane spanning domains which we named IM1 and IM2. Whereas IM1 was characterized in a previous study from our lab (Horvath et al., J. Biol. Chem. 287 (2012) 3674455) no information about IM2 has been provided so far. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria we constructed Psd1p variants with deletions in the predicted membrane spanning domain. Deletion of the complete IM2 led to mislocalization of the protein to the matrix site and to decreased enzyme activity. Deletion of the N-terminal moiety of IM2 also led to mislocalization to the matrix site of mitochondria, but deletions at the C-terminal part of IM2 resulted in localization to the outer mitochondrial membrane and to a loss of enzyme activity. In conclusion we showed that correct integration into the inner mitochondrial membrane is essential for full functionality of the yeast Psd1p.As a side-project we performed in collaboration with T. Becker Freiburg, Germany, studies on the influence of phosphatidylcholine (PC) on the two protein translocases TIM 22 and TIM 23 of the inner mitochondrial membrane. We showed that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. The reason for this finding is that the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.