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

Control and hormone-treated (48 h) GH4C1 cells were pulse labeled for 4 min with [35S]sulfate and then subjected to subcellular fractionation according to the standard procedure. After velocity sucrose gradient centrifugation, the peak of [35S]sulfitte-labeled granins was found to have a different distribution across the gradient than in the case of PC12 cells, being located in fractions 2-5. Fractions 3+4 were pooled, diluted 1 :1, divided into aliquots (two per gradient), and centrifuged (23,000 g for 30 min) . The pellet containing [ 35S]sulfate-labeled TGN vesicles was then incubated in nonaggregative or aggregative milieu in the presence of 1% Triton X-100 and analyzed as described above for the standard procedure.