Figure 9.

Entry and storage of proinsulin in secretory granules of GH4C1 cells. (A) Multiple identical wells of cells were pulse labeled with ³⁵S-amino acids as in Figure 6. At different chase intervals, a 30-min secretion period (ending with the time shown below the gels) was collected under unstimulated (−) or stimulated (+) conditions. The media were immunoprecipitated and analyzed by Tricine–urea–SDS-PAGE and fluorography; the positions of insulin and proinsulin are marked, and a conversion intermediate migrates above proinsulin in unstimulated and stimulated media. (B) The bar graphs show quantitation of the data as arbitrary units with paired bars of unstimulated versus stimulated secretion as a function of chase time for each of the three species of labeled peptide in the fluorograph (C, conversion intermediate; P, proinsulin; I, insulin). At early chase times, proinsulin entry into the stimulus-dependent secretory pathway is clear, although its utility as a granule marker wanes as a function of chase time. In contrast, insulin behaves as an ideal granule marker from the moment it first appears.