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

The presence of the granins in the supernatant upon incubation of TGN vesicles with saponin in nonaggregative milieu reflected the release of the lumenal content from permeabilized vesicles rather than the solubilization of the membrane, for three reasons. First, the bulk of the proteins was recovered in the pellet both in the absence and presence of saponin (Fig. 1, right) . Second, this was also observed for the hsPG (Fig . 1, left) , which is membrane-associated in the TGN as will be shown below (see Fig . 4 A) . Third, most of the proteins found in the supernatant in the presence of saponin were also present in the supernatant in the absence of saponin ( Fig . 1, right) . The few proteins that specifically increased in the supernatant upon saponin permeabilization included, besides CgB and SgII, four major proteins (dots and triangles in Fig. 1, right) . As will be shown below (see Fig . 5 ), these were ERresident proteins present in ERderived Chanat and Hunner Aggregation-mediating Sorting of Secretory Proteins sulfate-labeled TGN vesicles were obtained from PC12 cells, permeabilized with saponin in nonaggregative (NA) or aggregative (A) milieu, centrifuged, and pellets (P) and supernatants (S) were analyzed by SDS-PAGE followed by protein staining (bottom) and fluorography (top). All steps were performed according to the standard procedure . (Top) Note thatthe loss ofCgB from the pellet in nonaggregative milieu and its recovery in the pellet inaggregative milieu were readily detectable when the original fluorogram was examined in front of a strong light source . (B) [35S]sulfatelabeled TGN vesicles were obtained from PC12cells according to the standard procedure, permeabilized with saponin at pH 7.4, centrifuged, and the supernatant containing released CgB and SgIIwas incubated in aggregative milieu . After centrifugation, the pellet (P) and supernatant (S) were analyzed by SDS-PAGE and fluorography. The [35S]sulfate-labeled material in the pellet is due to the incomplete removal of the permeabilized TGN vesicles in the first centrifugation. (C) A heat-stable protein fraction of VSS]sulfate-labeled PC12 cells containing CgB and SgII was incubated in nonaggregative (NA) or aggregative (A) milieu, centrifuged, and the pellets (P) and supernatants(S) were analyzed by SDS-PAGE and fluorography.