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Vol. 17, Issue 12, 5038-5052, December 2006

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Not All Secretory Granules Are Created Equal: Partitioning of Soluble Content Proteins

Jacqueline A. Sobota^*, Francesco Ferraro^*, Nils Bäck, Betty A. Eipper^*, and Richard E. Mains^*

*Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030-3401; and Department of Anatomy, Institute of Biomedicine, University of Helsinki, FIN-00014, Helsinki, Finland

Submitted July 24, 2006; Revised September 14, 2006; Accepted September 20, 2006
Monitoring Editor: Randy Schekman

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine -hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Richard E. Mains (mains{at}uchc.edu)

Abbreviations used: GFP, green fluorescent protein; TGN, trans-Golgi network; POMC, proopiomelanocortin; ACTH, adrenocorticotropic hormone; PHM, peptidylglycine -hydroxylating monooxygenase; PAM, peptidylglycine -amidating monooxygenase; NPY, neuropeptide Y; CSFM, complete serum-free medium; PC1, prohormone convertase 1; PMA, phorbol 12-myristate 13-acetate

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