The AP-1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor

Abstract

Immature secretory granules (ISGs) in endocrine and neuroendocrine cells have been shown by morphological techniques to be partially clathrin coated (Orci, L., M. Ravazzola, M. Amherdt, D. Lonyard, A. Perrelet. 1985a. Proc. Natl. Acad. Sci. USA. 82: 5385-5389; Tooze, J., and S.A. Tooze. 1986. J. Cell Biol. 103:839-850). The function, and composition, of this clathrin coat has remained an enigma. Here we demonstrate using three independent techniques that immature secretory granules isolated from the rat neuroendocrine cell line PC12 have clathrin coat components associated with their membrane. To study the nature of the coat association we have developed an assay whereby the binding of the AP-1 subunit γ-adaptin to ISGs was reconstituted by addition of rat or bovine brain cytosol. The amount of γ-adaptin bound to the ISGs was ATP independent and was increased fourfold by the addition of GTPγS. The level of exogenous γ-adaptin recruited to the ISG was similar to the level of γ-adaptin present on the ISG after isolation. Addition of myristoylated ARF1 peptide stimulated binding. Reconstitution of the assay using AP-1 adaptor complex and recombinant ARF1 provided further evidence that ARF is involved in γ-adaptin binding to ISGs; BFA inhibited this binding. Trypsin treatment and Tris-stripping of the ISGs suggest that additional soluble and membrane-associated components are required for γ- adaptin binding.