Structure and Inheritance of the endoplasmic reticulum

描述（由申请人提供）：在所有真核细胞中，内质网（ER）形成一个高度开孔的管状网络，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。我们采用遗传学的方法来理解酵母内质网的动态、结构和遗传机制。我们迄今为止的工作表明，内质网小管在细胞周期开始时从核膜形成，它们通过V型肌球蛋白（Myo4p）沿着肌动蛋白电缆被输送到芽中，它们被外囊复合物锚定在芽尖，然后沿着细胞皮层扩散形成皮质内质网。我们认为rtn1p是保守网状蛋白家族的一员，作为内质网膜上囊胞亚基Sec6p的受体，并且rtn1p还可以将内质网连接到细胞皮层。规划了四个具体目标：

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.