Molecular Genetics of Regulated Protein Delivery of the Plasma Membrane

描述(由申请人提供)：完整的膜蛋白的活性，如运输蛋白，通常由细胞内分选来调节。这种分选过程可以使转运体响应细胞内信号而被运送到质膜的速度发生快速变化，从而为细胞提供了一种改变其从细胞外环境中摄取小分子的能力以响应调控信号的方法。例如，GLUT4葡萄糖转运体被输送到脂肪和肌肉细胞的质膜上，以响应胰岛素，而这种受调控的转运缺陷被认为是非胰岛素依赖型糖尿病的根本原因。GLUT4细胞内分选的许多方面目前仍知之甚少。Kaiser博士的研究小组建议继续研究普通氨基酸渗透酶(Gap1)的调节分类，以响应生长介质中的氮源。通过研究模式生物酿酒酵母中的氨基酸渗透酶分选，将有可能阐明在晚期分泌途径中调节分选的机制。上一个资助期的工作表明，Gap1p分选由Gap1p蛋白从内体循环到质膜的速度控制，这一分选步骤由细胞内氨基酸的丰富程度控制。对Gap1p循环缺陷突变株的遗传筛选发现了一个与Gap1p相互作用的含有GTPase的复合体(GSE)，这表明GSE复合体可能形成了Gap1p运输的囊泡涂层的一部分。在这些发现的基础上，建议：(I)确定GSE复合体的其他成分，最终目的是在体外重建活性；(Ii)确定GSE复合体的GTPase成分的结构和功能，包括哺乳动物同源基因的功能研究；以及(Iii)确定氮源如何控制Gap1 p循环。我们发现，Gap1p被泛素修饰是从高尔基体到内体分类所必需的。该项目的最终目标是(Iv)了解泛素化是如何被调控的，并确定识别泛素标签所需的细胞成分。

DESCRIPTION (provided by applicant): The activities of integral membrane proteins, such as transport proteins, are often regulated by intracellular sorting. Such sorting processes can produce rapid changes in the rate that a transporter is delivered to the plasma membrane in response to an intracellular signal and thus provide a way for a cell to alter its capacity to take up small molecules from the extracellular environment in response to regulatory cues. For example, the GLUT4 glucose transporter is delivered to the plasma membrane of fat and muscle cells in response to insulin, and defects in this regulated trafficking are thought to be a root cause of non insulin-dependent diabetes. Many aspects of the intracellular sorting of GLUT4 remain poorly understood at this time. Dr. Kaiser's research group proposes to continue to study regulated sorting of general amino acid permease (Gap1) in response to the nitrogen source in the growth medium. By studying amino acid permease sorting in the model organism S. cerevisiae it will be possible to elucidate the mechanisms responsible for regulated sorting in the late secretory pathway. Work in the previous funding period has shown that Gap 1 p sorting is controlled by the rate at which Gap1 p protein can recycle from the endosome to the plasma membrane and that this sorting step is controlled by the abundance of intracellular amino acids. A genetic screen for mutants defective in Gap1 p recycling identified a GTPase containing complex (GSE) that interacts with Gap 1 p so as to suggest that the GSE complex may form part of a vesicle coat for Gap 1 p trafficking. Building on these findings the proposal is to: (i) identify additional components of the GSE complex with the ultimate aim of reconstituting activity in vitro, (ii) determine the structure and function of the GTPase component of the GSE complex, including functional studies of the mammalian orthologs, and (iii) determine how the nitrogen source controls Gap1 p recycling. We have found that Gap1 p modification by ubiquitin is necessary for sorting from the Golgi to endosome. A final aim of the project is (iv) to understand how ubiquitination is regulated and to identify the cellular components required for recognition of the ubiquitin tag.