Molecular Genetics of Regulated Protein Delivery of the Plasma Membrane

描述（由申请人提供）：整体膜蛋白的活性，如运输蛋白，通常由细胞内分选调节。这种分选过程可以使转运蛋白响应胞内信号传递到质膜的速率发生快速变化，从而为细胞提供了一种方法，可以根据调节提示改变其从胞外环境中吸收小分子的能力。例如，GLUT4葡萄糖转运蛋白在胰岛素的作用下被传递到脂肪和肌肉细胞的质膜，这种受调节的运输缺陷被认为是非胰岛素依赖型糖尿病的根本原因。目前，对GLUT4细胞内分选的许多方面仍知之甚少。Kaiser博士的研究小组建议继续研究一般氨基酸渗透酶（Gap1）对生长培养基中氮源的调节分选。通过研究模式生物酿酒酵母的氨基酸渗透酶分选，将有可能阐明在后期分泌途径中负责调节分选的机制。先前资助期的工作表明，Gap 1p分选是由gap1p蛋白从内核体再循环到质膜的速度控制的，而这一分选步骤是由细胞内氨基酸的丰度控制的。对gap1p再循环缺陷突变体的遗传筛选发现了一种含有GTPase的复合物（GSE），该复合物与Gap 1p相互作用，从而表明GSE复合物可能形成用于Gap 1p运输的囊泡外壳的一部分。在这些发现的基础上，我们的建议是：(1)确定GSE复合物的其他组分，最终目的是在体外重建活性；(2)确定GSE复合物的GTPase组分的结构和功能，包括哺乳动物同源物的功能研究；(3)确定氮源如何控制gap1p的回收。我们发现通过泛素修饰gap1p对于高尔基体到核内体的分选是必要的。该项目的最终目标是（iv）了解泛素化是如何调节的，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.