Expression of Regulated Secretory Proteins Is Sufficient to Generate Granule-like Structures in Constitutively Secreting Cells

• Published in: The Journal of biological chemistry Vol. 279; no. 19; pp. 20242 - 20249
• Main Authors: Beuret, Nicole, Stettler, Hansruedi, Renold, Anja, Rutishauser, Jonas, Spiess, Martin
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 07-05-2004
• Subjects: Animals; Calcimycin - pharmacology; Cell Line; CHO Cells; Chromogranins - metabolism; COS Cells; Cricetinae; DNA, Complementary - metabolism; Dogs; Endoplasmic Reticulum - metabolism; Endosomes - metabolism; Glycoproteins - metabolism; Golgi Apparatus - metabolism; Humans; Ionophores - pharmacology; Kinetics; Lysosomes - metabolism; Membrane Glycoproteins; Membrane Proteins - metabolism; Mice; Microscopy, Electron; Microscopy, Fluorescence; NIH 3T3 Cells; Plasmids - metabolism; Proteins - metabolism; Secretory Vesicles - metabolism; Time Factors; trans-Golgi Network - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M310613200

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine,
neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin,
pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive
secretory protein α 1 -protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast
cells. The accumulations were observed in 30–70% of the transfected cells expressing the pro-hormones and for virtually all
of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated
secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy.
They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi
network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed,
they were frequently found in the same granules, whereas α 1 -protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated
from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and
chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The
results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules
in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.