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管状网络，分布在整个细胞体积中，通常位于质膜下方。这种分布可能对钙信号和其他重要的细胞功能至关重要。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亚基Sec6p的受体，Rtn1p还起到连接内质网与细胞皮质的作用。计划了四个具体目标： 1)我们将分析Rtn1p与胞囊的相互作用，确定Rtn1p在内质网膜内的拓扑结构以及连接胞囊与Rtn1p的特定相互作用。 2)我们将确定Rtn1p是否沿着皮质与Tcb3蛋白结合，这种相互作用是否支持Rtn1p的独特定位以及ER与细胞皮质的紧密联系。 3)我们将确定Rtn1p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)我们将表达或微量注射哺乳动物网状蛋白的显性负性等位基因，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的作用及其相互作用是否保守。

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.