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管状网络，遍布整个细胞体积，通常位于质膜下方。这种分布可能对钙信号传导和其他重要的细胞功能至关重要。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Myo4p沿着沿着肌动蛋白电缆被递送到芽中，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，Rtn1 p，保守的reticulon家族的蛋白质的成员，作为受体的外囊亚基Sec6p的ER膜上的功能，Rtn1 p也用于连接ER的细胞皮质。计划实现四个具体目标： 1)我们将分析Rtn1p与外囊的相互作用，定义ER膜内Rtn1p的拓扑结构以及将外囊与Rtn1p联系起来的特定相互作用。 2)我们将确定Rtn1 p是否与Tcb 3蛋白沿着皮层结合，以及这种相互作用是否是Rtn1 p独特定位以及ER与细胞皮层紧密联系的基础。 3)我们将确定Rtn1p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)我们将表达或微量注射哺乳动物reticulon蛋白的显性负等位基因，并利用RNAi方法抑制这四种哺乳动物RTN蛋白的功能。表型分析将确定Rtn1p及其相互作用的作用是否保守。

DESCRIPTION (provided by applicant): In all eukarotic cells, the endoplasmic reticulum (ER) forms a highly fenestrated tubular network that spreads throughout the volume of the cell, often lying just below the plasma membrane. This distribution may be critical for calcium signalling and other important cellular functions. 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 subunit Sec6p on the ER membrane and that Rtn1 p also serves to link the ER to the cell cortex. Four specific aims are planned: 1) We will analyze the interaction of Rtn1p with the exocyst, defining the topology of Rtn1p within the ER membrane and the specific interactions that link the exocyst to Rtn1p. 2) We will determine if Rtn1p binds to the Tcb3 protein along the cortex and if this interaction underlies the unique localization of Rtn1p as well as the tight association of the ER with the cell cortex. 3) We will determine if Rtn1p 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 express or microinject dominant negative alleles of mammalian reticulon proteins as well as make use of RNAi methodology to inhibit the function of the four mammalian RTN proteins. Phenotypic analysis will determine if the roles of Rtn1p and its interactions are conserved.