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

网格蛋白包被参与溶酶体生物发生的各个方面。网格蛋白与异四聚体接头蛋白（APs）结合。虽然AP-2在胞吞作用中起作用，但无处不在表达的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从细胞质池中被招募到特定的膜结构域，在那里它们与货物跨膜蛋白的细胞质结构域中的分选信号相互作用，从而将这些成分分离成运输中间体。APs如何在这些膜结构域上稳定在很大程度上是未知的。利用体外系统重现蛋白质分选的保真度，我们最近发现AP-1A和AP-3与合成膜的结合需要几种成分：ARF-1 GTPase，选定的跨膜蛋白的细胞质结构域和特定的磷酸肌苷。胞质结构域决定了AP-1A或AP-3的选择性相互作用，在PI-4P或PI-3P存在时，AP-1A或AP-3的结合分别增加了两倍。因此，我们的研究结果表明，多个结合位点及其比较化，如发生在反式高尔基网络上的PI-4P和早期核内体上的PI-3P，可能解释了AP-1A和AP-3在特定膜结构域上的选择性稳定。这个建议是双重的。首先，我们想说明PI-3P在体内AP-3与内体膜相互作用中的作用，并使用基于RNA干扰和功能分选试验的策略确定负责其产生的PI-3激酶。其次，我们的体外重构实验允许我们概括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细胞质蛋白，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.