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

The preceding data confirmed that a signal peptide was sufficient to effect translocation of globin into the ER in vivo but not for globin secretion. To determine if a propeptide could mediate secretion, GH3PRO-GLO cells were pulselabeled for 15 min with [35S]methionine and chased for up to 2 h (Fig. 2) . During the 15-min pulse, a prominent globinimrnunoreactive polypeptide migrating with the predicted molecular weight for PRO-GLO lacking a signal peptide (Mr 25,000) was detected intracellularly (Fig. 2, lane I, top arrow) . A second intracellular, globin-specific polypeptide (Mr 15,000) was detected between 15 and 30 min of chase (middle arrow; Fig. 2, lanes 2 and 3) . The appearance of this molecule, which had an electrophoretic mobility slightly slower than authentic c¢-globin (Fig. 2, bottom arrow) , was consistent with PRO-GLO being proteolytically processed at the single ArgLys site in proSRIF generating "mature" t~-globin: a-globin with four additional NH2-tenninal amino acids (see Fig. 1 ). Cleavage following the ArgLys was subsequently confirmed by partial NH2-terminal sequencing (see below and Fig. 6 ). With prolonged chase times, the level of Mr 15,000 globin increased with a concomitant reduction of the PRO-GLO polypeptide (Fig. 2, lanes 2-5) suggesting a precursor product relationship. Some unprocessed Mr 25,000 PRO-GLO was rapidly secreted (Fig. 2, lanes 7-10) but most significantly, processed mature Mr 15,000 globin was also secreted into the medium (Fig. 2, lanes 8-10, and Fig. 5 ).