The EARP Complex and its Interactor EIPR-1 are Required for 2 Cargo Sorting to Dense-Core Vesicles 3 4 Short title : The EARP Complex Controls Dense-Core-Vesicle Biogenesis

15 16. CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not. Abstract 17 18 The dense-core vesicle is a secretory organelle that mediates the regulated release of 19 peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the 20 trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is 21 formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle 22 biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants 23 defective in dense-core vesicle function. We previously reported the identification of two 24 conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core 25 vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-26 core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated 27 recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle 28 cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle 29 cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate 30 that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic 31 approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP 32 complex. Our data suggest that EIPR-1 is a new component of the EARP complex and that 33 dense-core vesicle cargo sorting depends on the EARP-dependent retrieval of cargo from an 34 endosomal sorting compartment. 35 36. CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not. Author Summary 37 Animal cells package and store many important signaling molecules in specialized 38 compartments called dense-core vesicles. Molecules stored in dense-core vesicles include 39 peptide hormones like insulin and small molecule neurotransmitters like dopamine. Defects in the 40 release of these compounds can lead to a wide range of metabolic and mental disorders in 41 humans, including diabetes, depression, and drug addiction. However, it is not well understood 42 how dense-core vesicles are formed in cells and package the appropriate molecules. Here we use 43 a genetic screen in the microscopic worm C. elegans to identify proteins that are important for 44 early steps in the generation of dense-core vesicles, such as …