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

Permeabilization of membrane vesicles present in the TGN preparation with saponin in nonaggregative milieu resulted in the release of several proteins, including four major polypeptides of 99, 78, 60, and 54 kD (Fig. 1 , right, dots and tri-Chanat and Hunner Aggregation-mediating Sorting of Secretory Proteins Figure 5 . ER resident proteins are released from saponin-permeabilized membrane vesicles in nonaggregative as well as aggregative milieu . (PNS) The membrane vesicles in a postnuclear supernatant (PNS) obtained from unlabeled PC12 cells were pelleted by centrifugation and incubated in the absence (-) or presence (+) of saponin in nonaggregative (NA) milieu . After centrifugation, pellets (P) and supernatants (S) were analyzed by SDS-PAGE followed by immunoblotting, using antisera against BiP (RaKSEK) or PDI (RaKAVK) followed by [ 115 I]protein A . Dots and triangles indicate proteins recognized by the RaKSEK and RctKAVK antiserum, respectively, which are also indicated in Fig. 1 . (Velocity gradient) Fractions 8-10 of the velocity gradient were obtained from unlabeled 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 immunoblotting using the antisera RaKSEK or RaKAVK and immunoperoxidase .