Role of lipid transfer proteins and membrane contact sites for intramitochondrial lipid trafficking

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Lipid transport in cells occurs in form of vesicles or in a non-vesicular manner. Recent evidence suggests that lipid trafficking in mitochondria involves dedicated lipid transfer proteins (LTPs) and is dependent on a close apposition of cellular membranes. We have identified novel LTPs that mediate the transport of phosphatidic acid across the intermembrane space of yeast and human mitochondria to allow cardiolipin biosynthesis in the mitochondrial inner membrane. These LTPs comprise heterodimeric complexes, which are composed of Ups1 (or PRELID1 in human) and Mdm35 (or TRIAP1 in human). The LTPs comprise a highly conserved protein family with several homologues being present in all eukaryotic cells, but the function of other members of the protein family remains enigmatic. The maintenance of the lipid composition of mitochondrial membranes also depends on membrane contact sites between the endoplasmic reticulum (ER) and the mitochondrial outer membrane and between both mitochondrial membranes. However, how these membrane contacts contribute to the trafficking of various lipids and how they cooperate with LTPs is only poorly understood. In the present project, we will define the role of LTPs and membrane contact sites for lipid trafficking in mitochondria and examine the function of different members of the Ups/PRELI family of LTPs. A focus of research will be on the role of Ups2 for the biogenesis of the major membrane constituent phosphatidylethanolamine (PE). We will define the role of Ups2 in PE biogenesis using biochemical and cell biological approaches in vivo. Purified Ups2-Mdm35 complexes will be used to examine a putative function of Ups2 as LTP and, if applicable, determine the lipid specificity of the LTP. These experiments will be complemented by studies on membrane tethering complexes, in order to unravel a function of contacts between both mitochondrial membranes during PE biogenesis. Moreover, we will analyze how phosphatidylglycerol is transported from the ER to the mitochondrial inner membrane for cardiolipin synthesis. We will assess the role of membrane tethering complexes for PG transport and perform a genome-wide genetic screen in yeast to identify novel proteins involved in lipid transport from the ER to the inner membrane. Finally, we will employ photoactivatable and clickable phospholipid derivatives to monitor lipid import into mitochondria and to identify novel lipid-protein interactions that occur during lipid transfer and synthesis in mitochondria. These studies will provide new insights into the mechanisms of lipid trafficking in mitochondria and will address issues of general cell biological relevance related to the role of LTPs and membrane contact sites.