Structure and Inheritance of the endoplasmic reticulum

描述(申请人提供)：在所有真核细胞中，内质网(ER)形成一个高度开窗的网络，分布在整个细胞体积中，通常位于质膜下方。我们采用了遗传学的方法来理解酵母中ER的动态、结构和遗传的机制。到目前为止，我们的工作表明，内质网小管在细胞周期开始时由核膜形成，它们由V型肌球蛋白Myo4p沿着肌动蛋白电缆传递到芽中，由外囊复合体锚定在芽尖，然后沿细胞皮质扩散形成皮质内质网。我们认为，Rtn1p是保守的网状蛋白家族中的一员，它是内质网膜上外囊的受体，Rtnlp还起到将内质网连接到细胞皮质的作用。我们进一步提出，麦角甾醇和鞘磷脂在控制细胞皮质ER遗传机制的一个关键组成部分的流动性或活性方面发挥着重要作用。计划有五个具体目标：1)我们将分析Rtn1p与胞囊的相互作用，确定Rtnlp在内质网膜内的拓扑结构，以及连接胞囊与Rtnlp的特定蛋白质-蛋白质相互作用。2)我们将确定Rtnlp是否与皮质上的蛋白质结合，这种相互作用是否支持Rtnlp的独特定位以及ER与细胞皮质的紧密联系。3)我们将确定Rtnlp是否在所有真核生物中形成ER的有窗网状结构特征中发挥作用，以及它是否以这种作用与ER膜的其他成分相互作用。4)我们将利用我们在酵母中分析Rtnlp的信息来构建哺乳动物网状蛋白的显性负等位基因。表型分析将确定Rtn1p的作用是否保守。5)我们将确定麦角甾醇和鞘磷脂是否对建立内质网结构、动力学和遗传的机械的皮质成分的定位、移动性或活性至关重要。

DESCRIPTION (provided by applicant): In all eukarotic cells, the endoplasmic reticulum (ER) forms a highly fenestrated network that spreads throughout the volume of the cell, often lying just below the plasma membrane. We have taken a genetic approach towards understanding the mechanism of ER dynamics, structure and inheritance in yeast. Our work to date shows that ER tubules form from the nuclear envelope at the start of the cell cycle, they are delivered into the bud along actin cables by the type V myosin, Myo4p, they are anchored at the bud tip by the exocyst complex and then spread along the cell cortex to form the cortical ER. We propose that Rtn1 p, a member of the conserved reticulon family of proteins, functions as the receptor for the exocyst on the ER membrane and that Rtnlp also serves to link the ER to the cell cortex. We further propose that ergosterol and sphingolipids play an important role controlling the mobility or activity of a key component of the ER inheritance machinery at the cell cortex. Five specific aims are planned: 1) We will analyze the interaction of Rtn1 p with the exocyst, defining the topology of Rtnlp within the ER membrane and the specific protein-protein interactions that link the exocyst to Rtnlp. 2) We will determine if Rtnlp binds to a protein along the cortex and if this interaction underlies the unique localization of Rtnlp as well as the tight association of the ER with the cell cortex. 3) We will determine if Rtnlp plays a role in forming the fenestrated reticulum characteristic of the ER in all eukaryotes and if it interacts with other components of the ER membrane in this capacity. 4) We will use the information from our analysis of Rtnlp in yeast to construct dominant negative alleles of mammalian reticulon proteins. Phenotypic analysis will determine if the roles of Rtn1 p are conserved. 5) We will determine if ergosterol and sphingolipids are critical for the localization, mobility or activity of a cortical component of the machinery that establishes the structure, dynamics and inheritance of the ER.