Characterization of granule-like structures in non-endocrine cells

All eukaryotic cells have transport pathways to constitutively secrete proteins. Endocrine, neuroendocrine, and exocrine cells in addition have a regulated secretory pathway that serves the controlled release of hormones, neuropeptides or digestive enzymes. In the transGolgi network (TGN) precursors of regulated secretory proteins are segregated and packaged in a concentrated form in secretory granules where they are processed and activated before release in response to an extracellular stimulus by fusion with the plasma membrane. Little is known about the machinery involved in the generation of secretory granules. The current models propose that luminal proteins interact with transmembrane receptors and/or that cargo proteins form insoluble aggregates due to milieu acidification resulting in membrane association and formation of a secretory granule. We discovered that even in non-endocrine cells the expression of regulated cargo proteins is sufficient to induce granule-like structures. We showed that cargo proteins accumulate in the TGN where they are sorted into membrane vesicles. These structures did not colocalize with organelle markers for the endoplasmic reticulum, the Golgi, the TGN, lysosomes, and endosomes. The helper cargo proteins secretogranin II and chromogranin B were stored intracellularly, and their secretion could be stimulated by addition of a calcium ionophore. Considerable differences were observed in efficiency of induction and in morphology of granule-like structures depending on the expressed cargo protein. Granins were more efficient, capable to sort other cargo proteins, and modulate the size of granule-like structures. Some accessory proteins such as the proprotein convertases PC3 and PC6A but also the IP3-R/Ca2+ channel sorted into granule-like structures when coexpressed with secretogranin II. The data suggest that secretory granule formation is a self-assembly process which is optimized by an endocrine-specific machinery. Carboxypeptidase E was proposed to function as a sorting receptor with an unconventional transmembrane anchor similar to PC2 and PC3. The topology of theses proteins is particularly important for granule sorting and association with putative interaction partners. A systematic analysis showed that PC3 is a luminal protein suggesting that granule targeting of PCs and CPE is not achieved by direct interaction with cytosolic proteins.