Regulation of Phospholipid Synthesis by the Master Transcription Factor Opi1

Opi1是一种两亲性转录抑制因子，调节磷脂的合成。磷脂酸(PA)是一种磷脂生物合成中间体，其在内质网(ER)中的水平调节Opi1ER的结合。一旦从内质网释放，Opi1进入细胞核，在那里它扮演着大量磷脂生物合成基因的转录抑制因子的角色。目的1.确定Opi1 LZ结构域在膜结合、定位和活性中的作用。我们进行了一项新的生化遗传学筛选，并确定了两亲性蛋白Opi1的独特等位基因，这些等位基因具有结构性活性。在我们分离的六个新的Opi1突变体中，有五个位于LZ结构域，而一个位于LZ结构域和相邻的PA结合区的交界处。我们认为这种串联结构域阵列可以作为膜状态的换能器来获得抑制活性。我们将使用纯化的Opi1来确定Opi1 LZ突变体与膜的亲和力，这种亲和力依赖于PA。还将在体内评估LZ区域的作用，以解决LZ在磷脂生物合成基因转录和内质网膜扩张中的膜结合和调节作用。目的2.确定Opi1 LZ结构域在Opi1自我相互作用中的作用。我们假设Opi1的LZ区介导了Opi1-Opi1的相互作用，Opi1二聚化的增加将驱动Opi1对磷脂生物合成基因的转录抑制。使用纯化的Opi1和我们的LZ突变体组合，我们将评估蛋白质二聚化和LZ在介导这种相互作用中的作用。我们还将使用已知的生长条件滴度Opi1膜相互作用来解决体内Opi1二聚化如何调节磷脂生物合成基因转录和内质网膜扩张。目的3.Opi1与Hac1相互作用以共同协调膜合成与UPR。我们推测Opi1可以通过与含有LZ的Hac1蛋白相互作用，将磷脂合成与UPR结合起来。HAC1是在ER合成的，以响应安装UPR的需要。我们已经成功地将Opi1与Hac1进行了免疫共沉淀。我们将使用纯化的蛋白质和蛋白质下拉列表来确定Opi1和Hac1是否相互作用，并使用我们的Opi1LZ突变体来评估LZ在这种相互作用中的作用。我们将继续进一步定位Opi1-Hac1相互作用结构域，并确定这种相互作用是否对UPR和Opi1介导的磷脂生物合成和内质网膜扩张是必要的。*这项研究项目的完成将增加我们对细胞如何感知和协调磷脂合成及其与UPR整合的了解。这将是我们对细胞生物学理解的重大进步，我们预计细胞生物学将成为本科生物化学教科书中的流行元素。

Opi1 is an amphipathic transcriptional repressor that regulates phospholipid synthesis. Phosphatidic acid (PA) is a phospholipid biosynthetic intermediate whose level in the endoplasmic reticulum (ER) regulate Opi1 ER binding. Upon release from the ER Opi1 enters the nucleus where is acts as a transcriptional repressor of a plethora of phospholipid biosynthetic genes.******Aim 1. Determining the Role of the Opi1 LZ Domain in Membrane binding, Localization, and Activity. We have performed a novel biochemical-genetics screen and identified unique alleles of the amphipathic protein Opi1 that are constitutively active. Five of the six new Opi1 mutants we isolated reside in the LZ domain, while one is at the junction of the LZ domain and the adjacent PA binding region. We propose that this tandem domain array could act as a transducer of membrane status into acquisition of repressing activity. We will use purified Opi1 to determine the affinity of the Opi1 LZ mutants for membranes in a PA dependent manner. The role of the LZ region will also be assessed in vivo to address the role of the LZ on membrane binding and regulation of phospholipid biosynthetic gene transcription and ER membrane expansion.******Aim 2. Determining the Role of the Opi1 LZ Domain in Opi1 Self-interaction. We hypothesize that the LZ region of Opi1 mediates Opi1-Opi1 interaction, and increased Opi1 dimerization will drive Opi1 transcriptional repression of phospholipid biosynthetic genes. Using purified Opi1 and our battery of LZ mutants, we will assess protein dimerization and the role of the LZ in mediating this interaction. We will also use growth conditions known to titer Opi1 membrane interaction to address Opi1 dimerization in vivo how dimerization regulates phospholipid biosynthetic gene transcription and ER membrane expansion.******Aim 3. Opi1 Interaction with Hac1 to Co-coordinate Membrane Synthesis with the UPR. We hypothesized that Opi1 could integrate phospholipid synthesis with the UPR via interaction with the LZ containing protein Hac1. Hac1 is synthesized at the ER in response to the need to mount an UPR. We have successfully co-immunoprecipitated Opi1 with Hac1. We will to determine if Opi1 and Hac1 interact using purified proteins and protein pull-downs, using our Opi1LZ mutants to assess the role of the LZ in this interaction. We will go on to further map the Opi1-Hac1 interaction domain and determine if this interaction is necessary for the UPR and Opi1 mediated phospholipid biosynthesis and ER membrane expansion. ******Completion of this research project will increase our knowledge on how how cells sense and coordinate phospholipid synthesis and its integration with the UPR. This would be a major advance in our understanding of cell biology that we expect would become elements prevalent in undergraduate biochemistry textbooks.