Membrane traffic is essential in eukaryotes as it is the process by which proteins are secreted from cells, organelle integrity is maintained, and both transmembrane proteins and lipids are transported within compartments of the cell. Many of the prevalent modern human diseases are now understood to be impacted or even wholly caused by defects or changes in membrane traffic;including cystic fibrosis, Alzheimer's disease, Parkinson's disease, and cancers. Substantial progress has been made in the last 25 years in our understanding of the roles played by specific proteins and lipids in membrane traffic. However, our molecular models remain rather crude in several areas and as the number of components identified at each step has soared a clear understanding of the sources of specificity and regulation in traffic has become muddled. This application focuses on the regulation of membrane traffic at one site, the late Golgi/trans-Golgi network (TGN), by specific protein cargos, Arf family regulatory GTPases, and the Arf dependent adaptors, Mints and GGAs. At least seven different Arf-dependent adaptors or complexes are directly involved in packaging cargo at the TGN and sorting to different destinations. We have developed reagents capable of the specific detection and depletion of each human Arf isoform and have used these to identify a number of novel sites of Arf action. Here we propose to use these and related reagents to focus on a single step of anterograde membrane traffic to address the growing complexity in traffic and the need for more detailed molecular models. This proposal has three specific aims. In the first we will determine the sources of specificity in Arf regulated traffic from the late Golgi/TGN by performing systematic tests in cultured mammalian cells of Arf isoforms required for recruitment and post-Golgi traffic of Mint, GGA, and AP-1 dependent carriers. We will test the hypothesis that the different Arf isoforms (Arf1, 3-5) act in pairs to directly recruit the different coat proteins or complexes to the late Golgi/TGN. In the second aim we will determine the sites of phosphorylation of Mint3 and Mint2 and the impact on cargo binding and localization to the TGN. In addition, we will test the model that GGA1 only work in combination with GGA2 and GGA3. We will test the hypotheses that protein phosphorylation of adaptors is a wide spread regulatory event in carrier biogenesis and that differences in regulation of homologous adaptors lead to differences in functions. And in aim #3 we will test the hypothesis that at least some cargo, e.g., APP, can exit the TGN using sorting machineries and that in doing so it leads to important differences in routing and processing. We will examine the traffic of APP and LR11 at the TGN and determine the sequence motifs involved in alternative APP exit. Membrane traffic is essential in eukaryotes as it is the process by which proteins are secreted from cells, organelle integrity is maintained, and both transmembrane proteins and lipids are transported within compartments of the cell. Public Health Relevance: This application focuses on the regulation of membrane traffic at one site, the late Golgi/trans-Golgi network (TGN), by specific protein cargos, Arf family regulatory GTPases, and the Arf dependent adaptors, Mints and GGAs. While of fundamental importance to our understanding of cell biology and cell signaling, our results have the potential to impact several of the most prevalent human diseases, including all that are now understood to be impacted or even wholly caused by defects or changes in membrane traffic;including cystic fibrosis, Alzheimer's disease, Parkinson's disease, and cancers.