Identification of a Chromogranin A Domain that Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic β-Cells Running title: New sorting partner of chromogranin A Key words: chromogranin, secretogranin, trans-Golgi network, secretory granule, sorting

Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH-and Ca 2 +-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were co-expressed in pituitary and pancreatic endocrine cell lines, while SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically co-localized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48-111) for SgIII mis-sorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214-373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localize with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, while other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells.