Structure and Inheritance of the endoplasmic reticulum

内质网的结构和遗传

• 批准号: 7030104
• 负责人: PETER Jay NOVICK
• 金额: $ 30.25万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030104
• 关键词: actins; binding proteins; binding sites; biochemical evolution; calcium flux; cell component structure /function; cell cycle; cell line; confocal scanning microscopy; cytoplasmic receptors; endoplasmic reticulum; fluorescence recovery after photobleaching; fungal proteins; immunoelectron microscopy; microinjections; myosins; nuclear membrane; protein binding; protein localization; yeasts

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.

描述（由申请人提供）：在所有真核细胞中，内质网（ER）形成高度有孔的管状网络，该网络遍布整个细胞体积，通常位于质膜正下方。这种分布对于钙信号传导和其他重要的细胞功能可能至关重要。我们采用遗传学方法来了解酵母内质网动态、结构和遗传的机制。迄今为止，我们的工作表明，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 沿着肌动蛋白缆输送到芽中，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ER。我们提出，Rtn1 p 是保守的网织蛋白家族的成员，其功能是作为 ER 膜上的外囊亚基 Sec6p 的受体，并且 Rtn1 p 还用于将 ER 连接到细胞皮层。规划了四个具体目标： 1）我们将分析 Rtn1p 与外囊的相互作用，定义内质网膜内 Rtn1p 的拓扑结构以及将外囊与 Rtn1p 连接的特定相互作用。 2) 我们将确定 Rtn1p 是否沿着皮质与 Tcb3 蛋白结合，以及这种相互作用是否是 Rtn1p 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 Rtn1p 是否在形成所有真核生物 ER 的有孔网状特征中发挥作用，以及它是否以这种能力与 ER 膜的其他成分相互作用。 4) 我们将表达或显微注射哺乳动物网状蛋白的显性失活等位基因，并利用RNAi方法来抑制四种哺乳动物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。