Molecular Studies of Selective Protein Transport

描述（由申请人提供）：网格蛋白包被囊泡（ccv）在将质膜蛋白分选入内吞途径以及在反式高尔基体网络（TGN）和内体之间分选蛋白质方面发挥重要作用。这些ccv介导的通路是真核细胞的基本保守元件;通路缺陷可导致遗传性人类疾病，并可能导致多基因疾病，如癌症，心脏病和阿尔茨海默病。此外，HIV等病原体利用这些途径感染细胞并避免免疫监视。这个项目的总体目标是了解正常细胞中ccv选择性蛋白质转运的分子基础，为理解缺陷如何导致疾病提供基础。为了实现这一目标，ccv介导的蛋白质转运已在酵母酿酒酵母中表征。在早期的研究中，我们表征了在TGN和内体之间的交通中起作用的网格蛋白衔接子网络。Gga蛋白和AP-1构成该网络的主要枢纽。在之前的资助期间，我们发现Gga蛋白和AP-1被依次招募到TGN以形成不同的网格蛋白外壳。这种衔接子进展的过程受TGN PI 4-激酶Pik 1 p产生的磷酸肌醇PI 4 P水平的调节，Pik 1 p似乎通过与Gga蛋白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，其中Gga蛋白和Pik 1 p之间的正反馈回路调节衔接子富集的ccv在TGN的逐步组装。我们对衔接子进展的研究揭示了以前未被重视的调控cCV形成的原则，并为在细胞器中组装功能专门化的包被囊泡提供了一种新的范例。因此，我们的研究结果开辟了独特的途径，以解决真核细胞膜交通的基本方面。遗传，生物化学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功能性地定义连续接头特异性ccv形成的过程，并将其与源自TGN的其他囊泡运输途径相关联。其次，互补的生化方法将用于表征调节衔接进展的分子基础，重点是测试Gga-Pik 1 p正反馈模型和定义衔接与PI 4P的相互作用。第三，前两个目标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。这些研究有望为TGN和内体之间通路中ccv形成的基本过程提供重要见解，从而有助于为理解这些过程在人类疾病中的作用奠定基础。

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.