Constitutive and basal secretion from the endocrine cell line, AtT-20

In the initial experiments on pathways of secretory protein sorting in AtT-20 cells, the regulated pathway was functionally defined by secretion of the ACTH hormone in response to a secretagogue while the constitutive pathway was defined by the externalization of a viral membrane protein, gp70, which did not require exposure to a secretagogue (Gumbiner and Kelly, 1982) . Unresolved in this work and in subsequent studies was whether constitutive secretion accounted for all unstimulated release, especially the slow release observed for "regulated" proteins, endogenous as well as transfected (Burgess et al., 1985; Moore and Kelly, 1985) . The AtT-20 variant was clearly lacking the regulated secretory pathway and all secretion from these cells was rapid and constitutive. The characteristics of secretion by the variant allow us to propose that there must be two pathways of unstimulated release in wild type cells: constitutive secretion, as described in our earlier work, and basal release from compartments that come after sorting into the regulated pathway. Since the regulated pathway is missing from the variant, basal release must also be missing. Basal release may be because of the fusion of mature regulated secretory granules with the plasma membrane in the absence of stimulation or the budding of vesicles from immature granules as proposed by yon Zastrow et al. (1987) . Whatever the mechanism of basal release, it accounts for a large fraction of the total secreted protein and has important consequences for the analysis of sorting in neuroendocrine cell lines. Thus measuring the ratio of stimulated to unstimulated release need not be a measure of sorting but a measure of what fraction of the material sorted into the regulated pathway that can be released by stimulation with secretagogues. Furthermore, the sorting index used by Moore and Kelly (1985) to measure protein sorting efficiencies, by ignoring basal release, underestimates the ability of AtT-20 cells to correctly sort proteins into the regulated pathway.