Molecular Genetics of Regulated Protein Delivery of the Plasma Membrane

描述（由申请人提供）：整合膜蛋白（如转运蛋白）的活性通常受细胞内分选的调节。这样的分选过程可以响应于细胞内信号而产生转运蛋白递送至质膜的速率的快速变化，从而为细胞提供一种改变其响应于调节线索而从细胞外环境摄取小分子的能力的方式。例如，GLUT 4葡萄糖转运蛋白响应于胰岛素被递送到脂肪和肌肉细胞的质膜，并且这种受调节的运输中的缺陷被认为是非胰岛素依赖型糖尿病的根本原因。GLUT 4的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一般氨基酸通透酶（Gap1）对生长培养基中氮源的调节分选。通过对模式生物S.酿酒酵母中，将有可能阐明机制负责调节排序在后期分泌途径。在之前的资助期间的工作表明，Gap 1 p分选是由Gap 1 p蛋白从内体再循环到质膜的速率控制的，并且该分选步骤是由细胞内氨基酸的丰度控制的。Gap1p再循环缺陷突变体的遗传筛选鉴定了与Gap 1p相互作用的含GTdR的复合物（GSE），从而表明GSE复合物可能形成Gap 1p运输的囊泡外壳的一部分。基于这些发现，建议是：（i）鉴定GSE复合物的额外组分，最终目的是体外重建活性，（ii）确定GSE复合物的GTdR组分的结构和功能，包括哺乳动物直向同源物的功能研究，和（iii）确定氮源如何控制Gaplp再循环。我们已经发现，Gap1 p的泛素修饰是必要的从高尔基体的内体排序。该项目的最终目的是（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.