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

Abstract

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. INTRODUCTIONThe cellular life of secretory proteins begins with the cotranslational translocation of their nascent polypeptide chains into the lumen of the endoplasmic reticulum (ER). These proteins then proceed in the anterograde direction, through the Golgi complex and into the trans-Golgi network (TGN), which is considered a "sorting station" for secretory proteins (Farquhar and Palade, 1981). At the TGN, vesicles destined for different subcellular compartments bud while acquiring their cargo. In all cell types, a "constitutive secretory pathway" delivers soluble and membrane secretory proteins necessary for housekeeping functions to the appropriate compartment (e.g., plasma membrane or endosomes/ lysosomes; Turner and Arvan, 2000). Besides this constitutive pathway, specialized cell types such as endocrine, exocrine, neuroendocrine cells, and neurons possess a "regulated pathway" in which specialized organelles, the secretory granules, store proteins for release in response to an incoming signal (Arvan and Castle, 1998). The soluble proteins found in constitutive vesicles and regulated secretory granules differ, and the processes responsible for this segregation are the object of intensive investigation.Two models, "sorting for entry" and "sorting by retention," both having experimental support, have been proposed to explain how sorting of soluble content proteins occurs (Arvan and Castle, 1998). The sorting for entry hypothesis postulates the existence of one or more "membrane receptors" able to selectivel...

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