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

Effect ofNonaggregative and Aggregative Milieu on Granins in the RER ofPC12 Cells PC12 cells were pulse labeled for 5 min with [3H]tyrosine and subjected to subcellular fractionation according to the standard procedure. After velocity sucrose gradient centrifugation, ['H]tyrosine-labeled granins were found throughout the gradient with a peak in fractions 2-5. Fractions 4+5, which contained less cytosolic proteins than fractions 2+3, were pooled, diluted 1:1, divided into aliquots (two per gradient), and centrifuged (23,000 g for 30 min) . The pellet containing [3H]tyrosine-labeled RER 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 .