Molecular Studies of Selective Protein Transport

描述(由申请人提供)：笼状蛋白包裹的囊泡(CCV)在将质膜蛋白分离到内吞途径以及在反高尔基网络(TGN)和内小体之间分离蛋白质方面发挥着重要作用。这些CCV介导的途径是真核细胞的基本保守成分；途径缺陷可能导致人类遗传性疾病，并可能导致癌症和心脏病等多基因疾病。该项目的总体目标是了解CCV在正常细胞中选择性蛋白运输的分子基础，为了解缺陷如何导致疾病提供基础。为了实现这一目标，我们在酿酒酵母中研究了CCV介导的蛋白质转运。在之前的资助期间，肌动蛋白相关蛋白Sla1p被确定为NPFX(1，2)D胞内分选信号的分选信号识别因子，Sla1p SHD1区域被定义为一个新的分选信号结合域。此外，还发现了SHD1在细胞壁合成的时间/空间调节中的生理作用。基于这些进展，遗传学、基因组学、生化、结构和细胞生物学方法将被应用于这些特定的目标：1)确定Sla1p在内吞作用中识别货物的机制；2)确定Sla1p在下调细胞壁合成中的作用；3)表征PKB/Akt激酶亚家族成员对SHD1活性的磷酸化调节；4)鉴定网状蛋白介导的内吞和TGN/内体运输途径的新成分。综上所述，这些研究有望在理解内吞作用中货物选择的分子基础和调控、选择性蛋白质识别在细胞生理学中扮演的角色以及作用于质膜、TGN和内小体的基于网状蛋白的运输机制的新组件的功能方面取得重大进展。

DESCRIPTION (provided by applicant): Clathrin-coated vesicles (ccv) play important roles in sorting plasma membrane proteins into the endocytic pathway and sorting proteins between the trans Golgi network (TGN) and endosomes. These ccv-mediated pathways are fundamental, conserved elements of eukaryotic cells; pathway defects can cause inherited human disorders and are likely to contribute to multigenic diseases such as cancer and heart disease. The overall goal of this project is to understand the molecular basis of selective protein transport by ccv in normal cells to provide a foundation for understanding how defects can lead to disease. Towards this goal ccv-mediated protein transport has been characterized in the yeast Saccharomyces cerevisiae. During the previous funding period the actin associated protein Sla1p was identified as a sorting signal recognition factor for NPFX(1,2)D endocytic sorting signals and the Sla1p SHD1 region was defined as a novel sorting signal binding domain. Also, a physiological role for SHD1 in the temporal/spacial regulation of cell wall synthesis was discovered. Based on these advances, genetic, genomic, biochemical, structural, and cell biological approaches will be applied to these specific aims: 1) define the mechanism of cargo recognition by Sla1p during endocytosis; 2) determine the role of Sla1p SHD1 in down-regulating cell wall synthesis; 3) characterize phospho-regulation of SHD1 activity by members of the PKB/Akt kinase subfamily; 4) identify new components of clathrin-mediated endocytic and TGN/endosome trafficking pathways. Together these studies are expected to provide significant advances in understanding the molecular basis and regulation of cargo selection in endocytosis, roles that selective protein recognition can play in cellular physiology, and functions of novel components of the clathrin-based transport machineries that act at the plasma membrane, TGN, and endosomes.