Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargoes including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting-protein EIPR-1 as proteins important for controlling levels of DCV cargoes in Caenorhabditis elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargoes such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.
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Downloads: 210History Submitted: 27 July 2018Revised: 17 October 2019Accepted: 8 November 2019
Information © 2020 Topalidou, Cattin-Ortolá, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
ACKNOWLEDGMENTS We thank Christopher Newgard, Ian Sweet, and Duk-Su Koh for the 832/13 cell line, with the support of the UW DRC Cell Function and Analysis Core; Suzanne Hoppins for the pBabe vector, platE cells, and protocol for lentiviral production and infection; Juan Bonifacino for the EARP and GARP subunit plasmids; Alex Merz and Sharon Tooze for antibodies; and Lloyd Fricker for antibodies and helpful discussions. J.C. was supported in part by a National Institutes of Health (NIH) Institutional Training Grant for Neurobiology (Grant no. T32 GM-007108). This work was supported by American Diabetes Association Grant no. 1-17-JDF-064 and by NIH Grant no. R01 GM-124035 to C.S.A., by a University of Washington Diabetes Research Center Pilot and Feasibility Award (NIH Grant no. P30 DK-017047), an Ellison Medical Foundation New Scholar Award, and by NIH Grant no. R00 MH-082109 and Grant no. R01 GM-121481 to M.A.
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