GENETICS OF PROTEIN DELIVERY TO 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。这种分选过程可以导致转运体响应细胞内信号而被运送到质膜的速度迅速变化，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。通过对酿酒酵母中氨基酸渗透酶分选的研究，将有可能利用一个发达的遗传有机体的全部力量来阐明在晚期分泌途径中调节分选的机制。在上一个资助期的工作已经确定了一大组控制Gap1排序的基因。这些基因包括直接参与Gap1膜转运的因子，例如用多聚泛素修饰Gap1所需的蛋白质，这是一种在高尔基体和内体隔室之间正确分选所需的标签。此外，还鉴定了为Gap1分类产生适当调控信号所需的基因。在这些发现的基础上，建议：(I)确定Gap1是如何泛素化的，并确定识别泛素标签所需的细胞成分；(Ii)结合遗传和生化方法来确定控制Gap1分选的基因产物的特征，并确定它们在分泌和内吞途径中的作用部位(S)；以及(Iii)确定氮源信号是如何产生的，并确定负责分选Gap1的膜运输机制如何解码这些信号。

DESCRIPTION (provided by applicant): The activities of integral membrane proteins, such as transport proteins, are often regulated by intracellular sorting. Such sorting process 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 S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to elucidate the mechanisms responsible for regulated sorting in the late secretory pathway. Work in the previous funding period has identified a large set of genes that control the sorting of Gap1. These genes include factors directly involved in the membrane trafficking of Gap1, such as proteins required to modify Gap1 with poly-ubiquitin, a tag found to be required for the proper sorting between Golgi and endosomal compartments. In addition, genes required to generate the proper regulatory signals for Gap1 sorting were also identified. Building on these findings the proposal is to: (i) determine how Gap1 is ubiquitinated and to identify the cellular components required for recognition of the ubiquitin tag, (ii) use a combination of genetic and biochemical methods to characterize the gene products that control Gap1 sorting and to determine their site(s) of action in the secretory and endocytic paths, and (iii) determine how nitrogen-derived signals are generated and determine how the membrane trafficking machinery responsible for sorting Gap1 decodes these signals.