The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells

We hypothesized that SIG-GLO was transported to lysosomes and degraded. To test this idea, GH3SIG-GLO cells were incubated with several concentrations of chloroquine . 3) , a weak base that neutralizes the intralysosomal acidic pH, thereby inactivating lysosomal hydrolases (23, 28) . At each chloroquine concentration (up to 150 #M), a-globin was degraded intracellularly and globin secretion was not observed (data not shown). This result suggested that the rapid turnover of the ot-globin molecules was not dependent on or occurring in an acidic compartment. The intracellular half-life of SIG-GLO was then determined by pulselabeling GH3SIG-GLO cells with [35S]methionine for 15 min and chasing for times up to 40 min; at each time point cells were lysed and the globin-immunoreactive products analyzed by SDS-PAGE (Fig. 4) . By 5 min of chase, >60% of u-globin had disappeared and by 40 min of chase globin was undetectable in the lysate; at no time point was globin detected in the medium. However, when cells were chased for 40 min at 16°C to prevent exit from the ER, 80-90% of the pulse-labeled ot-globin was still present intracellularly. Based on the rapid kinetics of the o~-globin disappearance, the temperature sensitivity, and the chloroquine data, it seemed unlikely that globin was transported to lysosomes for degradation.