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

细胞中的脂质转运以囊泡或非囊泡的形式发生。最近的证据表明，线粒体中的脂质运输涉及专门的脂质转移蛋白（LTPs），并依赖于细胞膜的紧密靠近。我们已经发现了新的LTPs，它们介导磷脂酸在酵母和人类线粒体的膜间空间的运输，从而允许在线粒体内膜中进行心磷脂的生物合成。这些LTPs包含异二聚体复合物，由Ups1（或人类PRELID1）和Mdm35（或人类TRIAP1）组成。LTPs包括一个高度保守的蛋白家族，在所有真核细胞中都有几个同源物，但该蛋白家族其他成员的功能仍然是谜。线粒体膜脂质组成的维持也取决于内质网（ER）和线粒体外膜之间以及两个线粒体膜之间的膜接触位点。然而，这些膜接触如何促进各种脂质的运输，以及它们如何与ltp合作，人们知之甚少。在本项目中，我们将定义LTPs和线粒体脂质运输的膜接触位点的作用，并检查LTPs的Ups/PRELI家族不同成员的功能。研究的重点将是Ups2在主要膜成分磷脂酰乙醇胺（PE）的生物发生中的作用。我们将使用生物化学和细胞生物学方法在体内定义Ups2在PE生物发生中的作用。纯化的Ups2- mdm35复合物将用于检测Ups2作为LTP的假定功能，并在适用的情况下确定LTP的脂质特异性。这些实验将通过膜系缚复合物的研究加以补充，以揭示PE生物发生过程中两种线粒体膜之间接触的功能。此外，我们将分析磷脂酰甘油如何从内质网转运到线粒体内膜以合成心磷脂。我们将评估膜系固复合物在PG转运中的作用，并在酵母中进行全基因组遗传筛选，以鉴定参与脂质从内质网转运到内膜的新蛋白。最后，我们将使用光激活和可点击的磷脂衍生物来监测脂质进入线粒体，并确定在线粒体脂质转移和合成过程中发生的新的脂质-蛋白相互作用。这些研究将为线粒体中脂质运输的机制提供新的见解，并将解决与ltp和膜接触位点的作用相关的一般细胞生物学问题。

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.