THE AP-1 ADAPTER COMPLEX BINDS TO IMMATURE SECRETORY GRANULES FROM PC12 CELLS, AND IS REGULATED BY ADP-RIBOSYLATION FACTOR

Immature secretory granules (ISGs) in endocrine and neuroendocrine cel ls have been shown by morphological techniques to be partially clathri n coated (Orci, L., M. Ravazzola, M. Amherdt, D. Lonvard, 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 compositio n, of this clathrin coat has remained an enigma. Here we demonstrate u sing three independent techniques that immature secretory granules iso lated from the rat neuroendocrine cell line PC12 have clathrin coat co mponents associated with their membrane. To study the nature of the co at association we have developed an assay whereby the binding of the A P-1 subunit gamma-adaptin to ISGs was reconstituted by addition of rat or bovine brain cytosol. The amount of gamma-adaptin bound to the ISG s was ATP independent and was increased fourfold by the addition of GT P gamma S. The level of exogenous gamma-adaptin recruited to the ISG w as similar to the level of gamma-adaptin present on the ISG after isol ation, Addition of myristoylated ARF1 peptide stimulated binding, Reco nstitution of the assay using AP-1 adaptor complex and recombinant ARF 1 provided further evidence that ARF is involved in gamma-adaptin bind ing to ISGs; BFA inhibited this binding. Trypsin treatment and Tris-st ripping of the ISGs suggest that additional soluble and membrane-assoc iated components are required for gamma-adaptin binding.