Protein targeting to endosomes/lysosomes II: protein networks regulating bidirectional transport between the trans-Golgi network and endosomes

网格蛋白被膜参与溶酶体生物发生的各个方面。网格蛋白与异源四聚体衔接蛋白（AP）缔合。虽然AP-2在胞吞作用中起作用，但广泛表达的AP-1A、AP-3和AP-4是跨膜蛋白从分泌途径转运到内体/溶酶体系统所必需的。AP从胞质池募集到特定的膜结构域上，在那里它们与货物跨膜蛋白的胞质结构域中存在的分选信号相互作用，从而将这些组分分离成转运中间体。AP如何稳定在这些膜结构域上在很大程度上是未知的。使用一个在体外系统，概括的保真度的蛋白质分选，我们最近表明，AP-1A和AP-3结合到合成膜需要几个组件：ARF-1 GT3，选定的跨膜蛋白和特定的磷酸肌醇的胞质结构域。胞质结构域决定AP-1A或AP-3的选择性相互作用，在PI-4P或PI-3 P存在下，AP-1A或AP-3的结合分别增加两倍。因此，我们的研究结果表明，多个结合位点和它们的compartimentalization，如已知的PI-4P发生在trans-Golgi网络和PI-3 P的早期内体，可能占AP-1A和AP-3与特定的膜结构域的选择性稳定。这项建议有两个方面。首先，我们想说明PI-3 P在体内的AP-3与内体膜的相互作用中的作用，并使用基于RNA干扰与功能分选分析相结合的策略来鉴定负责其产生的PI-3激酶。第二，我们的体外重建试验使我们能够概括AP-1或AP-3外壳的选择性形成，这促使我们使用蛋白质组学方法来鉴定与网格蛋白、AP-1A和AP-3一起形成外壳所需的胞质蛋白，然后表征它们的功能。

Clathrin-coats are involved in various aspects of lysosome biogenesis. Clathrin associates with hetero-tetrameric adaptor proteins (APs). While AP-2 functions in endocytosis, the ubiquitously expressed AP-1A, AP-3 and AP-4 are required for the transport of transmembrane proteins from the secretory pathway to the endosomal/lysosomal system. APs are recruited from a cytosolic pool onto specific membrane domains where they interact with sorting signals present in cytoplasmic domains of cargo transmembrane proteins, thereby segregating these components into transport intermediates. How APs are stabilized on these membrane domains is largely unknown. Using an in vitro system that recapitulates the fidelity of protein sorting, we have recently shown that AP-1A and AP-3 binding to synthetic membranes requires several components: the ARF-1 GTPase, cytoplasmic domains of selected transmembrane proteins and specific phosphoinositides. The cytoplasmic domains determine the selective interaction of AP-1A or AP-3, the binding of which are two fold increased in the presence of PI-4P or PI-3P, respectively. Thus, our results indicate that multiple binding sites and their compartimentalization, as known for PI-4P occurring on the trans-Golgi network and PI-3P on early endosomes, may account for selective stabilization of AP-1A and AP-3 with specific membrane domains. This proposal is two fold. First, we want to illustrate the role of PI-3P in the in vivo interaction of AP-3 with endosomal membranes and identify the PI-3 kinase responsible for its production using a strategy based on RNA interference coupled to functional sorting assays. Second, our in vitro reconstitution assay allowing us to recapitulate the selective formation of either AP-1 or AP-3 coats prompts us to use a proteomic approach to identify the cytosolic proteins required together with clathrin, AP-1A and AP-3 for coat formation and then characterize their function.