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

If the aggregative milieu reflects the milieu responsible for the aggregation of the granins in the TGN in vivo, it might be sufficient to induce the aggregation of the granins in the RER, a compartment in which these proteins are not aggregated as judged by the absence of electron-dense cores . We attempted to investigate this issue using saponin permeabilization of RER-derived vesicles prepared from PC12 cells pulse labeled for 5 min with ['H]tyrosine. However, for unknown reasons, the newly synthesized granins, in contrast to ER-resident proteins (see Fig . 5 ), were not released from saponin-permeabilized RER vesicles in nonaggregative milieu (data not shown) . Hence, instead of permeabilizing RER vesicles with saponin, we used Triton X-100 to investigate the effect of nonaggregative and aggregative milieu on newly synthesized granins. When RER vesicles, obtained from [3H]tyrosine-pulse-labeled PC12 cells after velocity gradient centrifugation, were incubated in 1 % Triton X-100 in nonaggregative milieu, two proteins of M 100,000 and M 85,000, corresponding to newly synthesized CgB and SgII, respectively, were recovered in the supernatant, whereas they were found in the pellet after incubation in aggregative milieu ( Fig . 6 A, right) . As in the case of TGN vesicles (see Fig. 1 A) , this behavior of the granins was quite specific since protein staining of the corresponding gel ( Fig . 6 A, left) showed that no other protein exhibited such a dramatic alteration in its distribution between pellet and supernatant in aggregative milieu as compared to nonaggregative milieu .