Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

Tb determine whether granins released from the lumen of the TUN in nonaggregative milieu would be converted to an aggregated state upon incubation in aggregative milieu, TGN vesicles obtained by the standard procedure were first permeabilized with saponin at pH 7.4 in a volume at least 100fold that ofthe packed vesicles, and centrifuged under conditions which pelleted most, but not all, of the permeabilized TGN membranes. When the granins obtained in the supernatant were incubated in aggregative milieu, they remained soluble, despite the presence of some TGN membranes (Fig. 2 B) . This indicated that aggregative milieu did not induce the aggregation of the granins after their release from the TGN, i.e., upon their dilution to a concentration at least 100fold lower than in the TGN lumen. This conclusion was supported by the observation that partially purified CgB and SgII remained soluble upon exposure to aggregative milieu at ti70 pg/ml (Fig. 2 C) , i.e., at a concentration at least two orders ofmagnitude below that in the TGN (estimated granin concentration >10 mg/ml) .