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）形成一个高度有孔的网络，遍布整个细胞体积，通常位于质膜下方。我们已经采取了遗传学的方法来了解ER动力学，结构和遗传在酵母中的机制。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，它们通过V型肌球蛋白Myo 4p沿着沿着肌动蛋白电缆被运送到芽中，它们通过外囊复合物锚定在芽尖，然后沿着细胞皮层扩散，形成皮层内质网。我们建议，Rtn 1 p，保守的reticulon家族的蛋白质的成员，作为ER膜上的外囊受体的功能，Rtnlp也用于连接ER的细胞皮质。我们进一步提出，麦角固醇和鞘脂在控制细胞皮质ER遗传机制的关键成分的流动性或活性方面发挥重要作用。我们计划了五个具体的目标：1）我们将分析Rtnlp与外囊的相互作用，定义Rtnlp在ER膜内的拓扑结构和连接外囊与Rtnlp的特定蛋白质-蛋白质相互作用。2)我们将确定Rtnlp是否与沿着皮质的蛋白质结合，以及这种相互作用是否是Rtnlp独特定位以及ER与细胞皮质紧密关联的基础。3)我们将确定Rtnlp是否在所有真核生物中形成内质网特征的有孔网中发挥作用，以及它是否与内质网膜的其他组分相互作用。4)我们将利用我们在酵母中的Rtnlp分析的信息来构建哺乳动物reticulon蛋白的显性负等位基因。表型分析将确定Rtn 1 p的作用是否保守。5)我们将确定麦角固醇和鞘脂是否对建立ER结构、动力学和遗传的机制的皮质成分的定位、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.