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、心脏病和阿尔茨海默病等多基因疾病。此外，艾滋病毒等病原体利用这些途径感染细胞，避免免疫监视。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。在早期的研究中，我们描述了一个在TGN和内体之间的交通中起作用的网状蛋白适配器网络。GGA蛋白和AP-1构成了这个网络的主要枢纽。在之前的资助期间，我们发现GGA蛋白和AP-1被顺序招募到TGN形成不同的笼状蛋白涂层。这一接头过程受TGN Pi4-Kinase Pik1p产生的磷脂酰肌醇PI4P水平的调节，该PI4P似乎通过与GGA蛋白的直接相互作用被招募到TGN。基于这些发现，我们提出了一个模型，在该模型中，GGA蛋白和Pik1p之间的正反馈环调节富含接头的CCV在TGN的渐进组装。我们对接头进程的研究揭示了以前未被认识到的CCV形成调节原理，并为在细胞器组装功能特化的被覆小泡提供了一种新的范例。因此，我们的发现为解决真核膜运输的基本方面开辟了独特的途径。遗传、生化和活细胞成像策略的组合将被应用于实现三个具体目标。首先，我们将应用活细胞成像和遗传策略来从功能上定义序列接头特异性CCV的形成过程，并将其与TGN发出的其他囊泡运输途径联系起来。其次，将使用互补的生化方法来表征调节适配器进程的分子基础，重点是测试GGA-Pik1p正反馈模型并定义与PI4P的适配器相互作用。第三，前两个目标中的方法将被扩展到评估保守的TGN/内体辅助因子在CCV形成中的功能。总之，这些研究有望为TGN和内小体之间的通路中CCV形成的基本过程提供重要的见解，从而有助于为理解这些过程在人类疾病中所起的作用奠定基础。

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, heart disease, and Alzheimer's disease. Also, pathogens such as HIV take advantage of these pathways to infect cells and avoid immune surveillance. 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. In earlier studies we characterized a network of clathrin adaptors that function in traffic between the TGN and endosomes. Gga proteins and AP-1 constitute major hubs in this network. During the previous funding period we discovered that Gga proteins and AP-1 are recruited sequentially to the TGN to form distinct clathrin coats. This process of adaptor progression is regulated by levels of the phosphoinositide PI4P that are generated by the TGN PI4-kinase Pik1p, which appears to be recruited to the TGN by direct interaction with Gga proteins. Based on these findings we have proposed a model in which a positive feedback loop between Gga proteins and Pik1p regulates progressive assembly of adaptor-enriched ccv at the TGN. Our studies of adaptor progression reveal previously unappreciated principles for regulation of ccv formation and offer a novel paradigm for assembly of functionally-specialized coated vesicles at an organelle. Thus, our findings have opened up unique avenues to address fundamental aspects of eukaryotic membrane traffic. A combination of genetic, biochemical, and live cell imaging strategies will be applied to achieve three specific aims. First, we will apply live cell imaging and genetic strategies to functionally define the process of sequential adaptor- specific ccv formation and relate it to other vesicle trafficking pathways emanating from the TGN. Second, complementary biochemical approaches will be used to characterize the molecular basis for regulation of adaptor progression with an emphasis on testing the Gga-Pik1p positive feedback model and defining adaptor interactions with PI4P. Third, approaches in the first two aims will be extended to assess the functions of conserved TGN/endosome accessory factors in ccv formation. Together these studies are expected to provide significant insights into the fundamental process of ccv formation in pathways between the TGN and endosomes, thereby helping to establish a foundation for understanding the roles these processes play in human disease.