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

We investigated the morphological correlate of the granin aggregates recovered after membrane permeabilization in aggregative milieu . Fractions enriched in immature secretory granules, identified by SgII pulse labeled with ['IS]sulfate and chased for 15 min, were obtained by differential centrifugation of a PC12 cell postnuclear supernatant in isotonic sucrose (Tooze et al., 1991) , incubated in various conditions, fixed, and analyzed by EM. Samples incubated in the absence of saponin were characterized by the presence of numerous (284/100 pmt) dense-cored structures (Fig. 3 , A-C) which had the typical morphological appearance of PC12 cell immature secretary granules (Tooze and Huttner, 1990) . Almost no (6/100 1mz) dense cores were found when membranes were permeabilized by saponin in nonaggregative milieu and fixed 5 min later (Fig. 3 , D-F). In contrast, after membrane permeabilization by saponin in aggregative milieu (Fig. 3, GI) , dense cores were observed at a frequency (197/100 ,um') that was -70% of that of the nonpermeabilized control. Interestingly, the dense cores recovered after saponin permeabilization in aggregative milieu appeared to be less electron-dense than those ofthe nonpermeabilized control. This may suggest that molecules other than the granins, which may not be retained in the aggregates aftermembrane permeabilization in aggregative milieu (e.g., ATP and catecholamines), contributed to the electron density of the cores. In conclusion, the recovery of dense cores after membrane permeabilization in aggregative, but not nonaggregative, milieu indicates that these are the morphological correlate of the granin aggregates observed biochemically under the former condition .