A signal sequence is sufficient for green fluorescent protein to be routedto regulated secretory granules

To investigate trafficking in neuroendocrine cells, green fluorescent prote in (GFP) tags were fused to various portions of the preproneuropeptide Y (N PY) precursor. Two neuroendocrine cell lines, AtT-20 corticotrope tumor cel ls and PC-12 pheochromocytoma cells, along with primary anterior pituitary cells, were examined. Expression of chimeric constructs did not disrupt tra fficking or regulated secretion of endogenous ACTH and prohormone convertas e 1 in AtT-20 cells. Western blot and immunocytochemical analyses demonstra ted that the chimeric constructs remained intact, as long as the Lys-Arg cl eavage site within preproNPY was deleted. GFP was stored in, and released f rom, regulated granules in cells expressing half of the NPY precursor fused to GFP, and also in cells in which only the signal sequence of preproNPY w as fused to GFP. Thus, in neuroendocrine cells, entering the lumen of the s ecretory pathway is sufficient to target GFP to regulated secretory granule s.