Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

In endocrine cells, prohormones and granins are segregated in the trans-Golgi network from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner upon specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures which resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of chromogranin A is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP or a short epitope tag was expressed in COS-1 fibroblast cells and analyzed by fluorescence and electron microscopy and pulse-chase labeling. Full-length chromogranin A as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that colocalized with coexpressed secretogranin II. These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.