Lipid transporters and lipid homeostasis at membrane contact sites

 描述(申请人提供)：在真核生物中，内质网(ER)和另一个细胞器紧密接触的膜接触部位(MCSs)在脂质稳态中起着关键作用，但在这些部位发生的过程在分子水平上仍然知之甚少。为了更好地了解接触部位的功能和发生在那里的活动的调节，我们的方法是表征驻留在这些部位的蛋白质机制，重点是内质网和质膜(PM)之间的接触。最近被认为是由MCSs介导的一个重要的脂质动态平衡过程是脂转移蛋白对脂类的非囊泡运输。我们将结合结构、生化和基于细胞的研究来表征ER-PM接触部位的脂类转运体，解决哪些蛋白质参与脂类转运，它们如何靶向这些部位，哪些脂类参与你的转运，它们如何识别它们的脂类配体，以及这些蛋白质可能如何调控这些蛋白质的转运。目的1重点研究TMEM24蛋白，它只存在于后生动物中，最近被发现是胰岛素分泌的调节因子。初步数据表明，TMEM24是一种内质网-PM接触者，存在一个与磷脂酰肌醇特异结合的脂结合模块 (PI)。在探讨TMEM24在接触部位活性的机制时，我们将检验这样的假设：TMEM24将PI从其在内质网中的合成位置转移到PM，在PM中PI可以快速转化为磷脂酰肌醇-4，5-二磷酸[PI(4，5)P2]。PI(4，5)P2是一种主要的PM信号转导脂质，对囊泡停靠和启动至关重要，我们认为TMEM24在分泌过程中的作用是供应PM PI(4，5)P2。在目标2中，我们将使用类似的方法来表征锚定在膜接触位点(LAM)家族的脂转移蛋白中的蛋白质，该家族在酵母中新近被发现是类固醇运输的关键，类固醇是PM的主要成分。我们将把对LAM家族的研究扩展到后生动物蛋白质，确定该家族中哪些成员是ER-PM接触的目标，探索它们是否起到与真菌相同的关键作用，并通过以下方式从结构和功能上剖析分子机制 它们的目标是ER-PM接触者，并将胆固醇输送到PM

 DESCRIPTION (provided by applicant): In eukaryotes, membrane contact sites (MCSs), at which the endoplasmic reticulum (ER) and another organelle come into close proximity, play key roles in lipid homeostasis, but the processes that occur at these sites remain poorly understood at the molecular level. Our approach to better understanding the function of contact sites and the regulation of activities that take place there is to characterize the protein machinery residen at such sites, focusing on contacts between the ER and the plasma membrane (PM). One process recently identified as important for lipid homeostasis as mediated by MCSs is the non- vesicular transport of lipids by lipid transfer proteins. We will combine structural, biochemical, and cell- based studies to characterize lipid transporters at ER-PM contact sites, addressing which proteins participate in lipid transfer, how they are targeted to these sites, which lipids thy transport, how they recognize their lipid ligands, and how transport by these proteins might be regulated. Aim 1 focuses on the protein TMEM24, present only in metazoans and recently identified as a regulator of insulin secretion. Preliminary data indicate TMEM24 as an ER-PM contact resident with a lipid-binding module that specifically associates with phosphatidylinositol (PI). In probing the mechanisms that underlie TMEM24 activity at contact sites, we will test the hypothesis that TMEM24 transports PI from its place of synthesis in the ER to the PM, where PI can be rapidly converted to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 is a major PM signaling lipid critical for vesicle docking and priming, and we propose that the role of TMEM24 in secretion is to supply PM PI(4,5)P2. In Aim 2, we will apply a similar approach to characterize proteins in the lipid transfer proteins anchored at membrane contact sites (LAM) family, newly identified in yeast as key for the transport of sterol, a major PM component. We will expand investigations of the LAM family to the metazoan proteins, identifying which members of this family are targeted to ER- PM contacts, probing whether they play the same critical role as in fungi, and structurally and functionally dissecting the molecular mechanisms by which they are targeted to ER-PM contacts and transport cholesterol to the PM.