Secretogranin II (SgII) is a protein specific to the matrix of the secretory granules in neurons and neuroendocrine cells. We have already demonstrated the precursor-product relationship between sulfated SgII and four N-terminal derived peptides in GH3B6 prolactin cells. In this study, we have investigated the subcellular compartment in which the cleavage of SgII is initiated by taking advantage of its tyrosine sulfation in the transGolgi network (TGN). In order to prevent export of radiosulfated SgII from the TGN, we used brefeldin A (BFA) as well as incubation at 20°C. BFA completely inhibited the cleavage of SgII when added immediately post-pulse. BFA added a few minutes post-pulse or after a 20°C incubation, however, permitted the cleavage of SgII in the presence of the drug. These SgII-derived peptides generated in the presence of BFA could not be released upon stimulation of the cells by either thyroliberin, a physiological secretagogue, or KCl. These results demonstrate that SgII can be cleaved in the TGN. They also evidence that the cleavage occurs in a distal compartment of the TGN different from the sulfation site. The transfer of SgII from the sulfation site to this distal compartment of the TGN involves BFA-sensitive membrane dynamics.Bioactive peptides and hormones secreted by neurons and neuroendocrine cells are synthesized as precursors that must undergo post-translational processing before their release. The different steps of this processing occur during the vectorial transport of the proteins from the rough endoplasmic reticulum, where they are synthesized, to the secretory granules, in which they are stored. Proteolytic processing of the precursors is one of the last of these post-translational modifications. Although it occurs mainly in the secretory granules, it can be initiated in the trans-Golgi network (TGN) 1 (reviewed in Ref. 1). Indeed, immunocytochemical (2-5) and biochemical studies (6 -9) have evidenced the cleavage of precursors in the TGN as well as in the secretory granules. In this study, we have investigated the intracellular cleavage site of secretogranin II (SgII) in GH3B6 prolactin cells.SgII is a member of the granin family (reviewed in Ref. 10).These proteins are specific to the dense core secretory granules of neuroendocrine cells and neurons (reviewed in Ref. 35 S]sulfate has already allowed the use of sulfated SgII as a marker of intracellular transport between the TGN and the plasma membrane via the dense core secretory granules (23-25). PC12 cells, however, lack the prohormone convertases PC1 and PC2, which are involved in the processing of SgII (26,27). Our experiments were performed in GH3B6 cells, a subclone of GH3 cells, which express PC2 but not PC1 (28). Cultivating these cells in the presence of insulin, 17-estradiol, and epidermal growth factor increases the number of secretory granules (29), the expression of PC2 (28) and the storage of SgII-derived peptides in the granules (19). GH3B6 cells therefore provide an ideal model for studying the proteolytic proce...