Synaptotagmin (Syt) IX is an essential determinant for protein sorting to secretory granules in mast cells.

The secretory granules (SGs) of secretory cells of the hematopoietic lineage, such as the mast cells, are lysosome-related organelles whose membrane proteins travel through the plasma membrane and the endocytic system. Therefore, a mechanism must exist to prevent proteins destined to recycling or to the trans-Golgi network (TGN) from reaching the SGs. We now show that synaptotagmin (Syt) IX, a Syt homologue that is required for recycling from the endocytic recycling compartment (ERC) in rat basophilic leukemia (RBL-2H3) cultured mast cells, is involved in segregating recycling proteins from the SGs. By using as a marker the recycling protein TGN38, which cycles between the TGN, plasma membrane, and the ERC, we show that knock-down of Syt IX results in mistargeting of HA-tagged TGN38 to the SGs. We further demonstrate that Syt IX binds directly the small GTPase ARF1 and associates with the clathrin adaptor complex AP-1. These results therefore implicate Syt IX as an essential factor for the correct sorting of SGs proteins. Moreover, they place Syt IX as part of the machinery that is involved in the formation of transport carriers that mediate SGs protein sorting.