Figure 7.

Relative storage of proinsulin and insulin in secretory granules of GH4C1 cells labeled to approach steady state. Different clones that either manufacture insulin (1-5 and 1-67) or do not manufacture insulin (Con) are shown. Cells were labeled as in Figure 5. Media collected during 3 initial unstimulated hours (middle panels) are shown as lanes marked 1, 2, and 3 for each clone. (As observed previously [Figure 5B], the 3-h period allows unstored peptides to drain through the intracellular transport pathway into the medium; this also enriches the cells for slow-turnover pools of labeled secretory protein.) Additional unstimulated and stimulated media at the end of the experiment are shown in each case in lanes 4 and Stim (panels at right). The total contents of labeled proinsulin and insulin produced by each clone were analyzed by immunoprecipitation in the gels shown at the far left. Quantitative analyses of the data for the proinsulin converters (PC1-expressing clones) and the nonconverter (Con) are shown in the bar graph at the far right: secretion of proinsulin is shown in the hatched bars, and secretion of insulin is shown in the closed bar. Conservation of cysteines and methionines between rat proinsulins and rat insulins allows the radioactivity in these bars to be directly additive. The stimulus-dependent secretion (stimulated minus unstimulated) is represented by square brackets in the bar graph. Note that all of the increase in stimulus-dependent release from the PC1-expressing clones is in the form of insulin.