Molecular Basis for Membrane Lipid Homeostasis

摘要 围绕不同细胞器的膜双层的脂质成分在两个方面都是独一无二的 结构脂和信号脂，如磷脂酰肌醇，赋予每个隔室不同的生化 以及其功能所固有的生物物理特性。在这份米拉提案中，我们解决了基本的和 细胞如何维持这些不同的脂质成分，即使在连续的 腔隙间的囊泡运输和脂类交换。我们的研究将集中在两个鲜为人知的 控制脂质稳态的机制：膜接触部位的脂质交换和脂类重塑 多功能磷脂酰肌醇激酶/磷酸酶复合体。膜接触部位，其中两个 细胞器密切相关，正在出现，在膜脂动力学和 动态平衡。为了发现在这些地点发生的过程及其分子基础，我们正在探索 哪些蛋白质定位在那里，它们的功能是什么，它们是如何以及何时在那里被招募的，以及它们的活性是如何 是受监管的。我们在下一个项目期间的研究将集中在VPS13和相关蛋白质上，这是我们的建议 初步数据，以组成一个新的脂类运输蛋白家族。这些研究有望获得令人振奋的新见解。 膜生物学，包括长期存在的线粒体和自噬如何 隔离膜既不连接到已建立的囊泡运输途径，也可能获得它们的 膜脂。膜接触部位也可以调节存在的肌醇磷脂种类的水平。 在不同的隔室，但由脂蛋白激酶和脂磷酸酶调节与外围相关 单个细胞器的膜双层可能在局部控制中起着更重要的作用。 磷脂酰肌醇水平。为了更好地了解肌醇磷脂动态平衡的机制，我们正在 表征这些可通过磷酸化可逆地相互转化肌醇磷脂物种的酶 以及它们的肌醇头基的去磷酸化。特别是，在下一个项目期，我们将重点关注如何 磷脂酰肌醇-(3，5)-二磷酸(PI(3，5)P2)的水平，它在糖尿病的生物学中起着核心作用 溶酶体/液泡，受PIKfyve复合体调控。PI(3，5)P2代谢的机制 都是难以捉摸的，部分原因是这个大会的复杂性，它至少包括三个不同的 蛋白质和拮抗脂蛋白激酶和脂磷酸酶活性。在体外研究这种复合体，分离 从活细胞中正在进行的许多过程中，将是理解PI(3，5)P2合成和 退化是单独调节的，并最终得到协调。对于这些项目，我们将利用我们的 在体外结构、生化和生物物理技术方面的专业知识，然后对提出的假设进行功能测试 通过良好的合作或与细胞生物学家同事协商。

Abstract The lipid composition of the membrane bilayer surrounding different cellular organelles is unique both in terms of structural lipids and signaling lipids like the phosphoinositides, lending each compartment distinct biochemical and biophysical characteristics intrinsic to its function. In this MIRA proposal, we address the fundamental and largely unexplored question of how cells maintain these distinct lipid compositions, even in light of continuous vesicle trafficking and lipid exchange between compartments. Our research will focus on two poorly understood mechanisms for controlling lipid homeostasis: lipid exchange at membrane contact sites and lipid remodeling by multi-functional phosphoinositide kinase/phosphatase complexes. Membrane contact sites, where two organelles come into close apposition, are emerging to play a critical role in membrane lipid dynamics and homeostasis. To discover the processes occurring at such sites and their molecular basis, we are exploring which proteins localize there, what their function is, how and when are they recruited there, and how their activity is regulated. Our studies in the next project period will focus on VPS13 and related proteins, suggested by our preliminary data to comprise a new family of lipid transport proteins. These studies promise exciting new insights into membrane biology, including for the long-standing questions of how mitochondria and the autophagosomal isolation membrane, neither connected to well-established vesicular trafficking pathways, may acquire their membrane lipids. Membrane contact sites can also modulate the levels of phosphoinositide lipid species present at different compartments, but regulation by lipid kinases and lipid phosphatases peripherally associated with the membrane bilayer of individual organelles likely plays a more significant role in controlling local phosphoinositide levels. To better understand the mechanisms governing phosphoinositide homeostasis, we are characterizing these enzymes, which reversibly interconvert phosphoinositide species via the phosphorylation and dephosphorylation of their inositol headgroups. In particular, in the next project period, we will focus on how levels of phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2), which plays a central role in the biology of the lysosome/vacuole, are regulated by the PIKfyve complex. The mechanisms underlying PI(3,5)P2 metabolism have been elusive, owing in part to the complexity of this assembly which comprises at least three different proteins and antagonistic lipid kinase and lipid phosphatase activities. Studying this complex in vitro, separate from the many processes ongoing in living cells, will be critical in understanding how PI(3,5)P2 synthesis and degradation are individually regulated and ultimately coordinated. For these projects, we will leverage our expertise in structural, biochemical, and biophysical techniques in vitro, then test arising hypotheses functionally via well-established collaborations or in consultation with cell biologist colleagues.