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

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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.