The EARP Complex and its Interactor EIPR-1 are Required for Cargo Sorting to Dense-Core Vesicles

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new component of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent retrieval of cargo from an endosomal sorting compartment.\n\nAuthor SummaryAnimal cells package and store many important signaling molecules in specialized compartments called dense-core vesicles. Molecules stored in dense-core vesicles include peptide hormones like insulin and small molecule neurotransmitters like dopamine. Defects in the release of these compounds can lead to a wide range of metabolic and mental disorders in humans, including diabetes, depression, and drug addiction. However, it is not well understood how dense-core vesicles are formed in cells and package the appropriate molecules. Here we use a genetic screen in the microscopic worm C. elegans to identify proteins that are important for early steps in the generation of dense-core vesicles, such as packaging the correct molecular cargos in the vesicles. We identify several factors that are conserved between worms and humans and point to a new role for a protein complex that had previously been shown to be important for controlling trafficking in other cellular compartments. The identification of this complex suggests new cellular trafficking events that may be important for the generation of dense-core vesicles.