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

[35S]sulfate-labeled TGN vesicles obtained according to the standard procedure were incubated with Triton X-100 in aggregative milieu and centrifuged. The pellet was then incubated in either aggregative or nonaggregative milieu, and recentrifuged . Upon incubation in nonaggregative milieu, most of the [33S]sulfatelabeled CgB and SgII (>80%) was recovered in the supernatant, whereas the majority ofboth granins (>80%) remained in an aggregated state when the aggregative milieu was maintained (data not shown) . Protein staining of the gel indicated The Journal of Cell Biology, Volume 115, 1991 Figure 6. Aggregative milieu allows the recovery of granin aggregates from detergent-treated RER vesicles of PC12 cells. PC12 cells were labeled for 5 min with [3H]tyrosine and processed according to the standard procedure except that fractions 4+5 of the velocity sucrose gradient were used . RER vesicles were incubated with Triton X-100 in nonaggregative (NA) or aggregative (A) milieu, centrifuged, and pellets (P) and supernatants (S) were analyzed by SDS-PAGE followed by protein staining (left) and fluorography (right) . Note that most of the granins detected by protein staining are derived from secretory granules also present in the fractions .