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

The state of aggregation of V5S]sulfate-labeled granins and GAG chains in the TUN of xyloside-treated PC12 cells upon incubation in nonaggregative or aggregative milieu was studied according to the standard procedure, with the following modifications for the GAG chains. After permeabilization of [35 S]sulfate-labeled TGN vesicles with 0.5 mg/ml saponin in nonaggregative or aggregative milieu and centrifugation (110,000 g for 15 min), the supernatant, which contained the GAG chains released from the TUN vesicles, was collected for analysis. The pelleted membranes were resuspended in 100 pl of 10 mM Tris-HCI, pH 7.4, 10 mM EDTA, 150 mM NaCl, incubated for 5 min at 100°C and centrifuged at 110,000 g for 15 min to obtain a heat-stable fraction containing the GAG chains . GAG chains in the supernatant and GAG chains in the heat-stable fraction of the pellet were then analyzed by SDS-PAGE followed by fluorography, except that the staining and destaining of the gel was omitted (see below) .