A conserved alpha-helix at the amino terminus of prosomatostatin serves asa sorting signal for the regulated secretory pathway

Mammalian prosomatostatin (PSST) contains the bioactive peptides SST-14 and SST-28 at the COOH-terminal end of the molecule and a putative sorting sig nal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH2-terminal segment of PSST consists of an amphipat hic cu-helix, which has been totally conserved throughout vertebrate evolut ion. We have analyzed the PSST-(3-15) region for sorting function by alanin e scanning and deletional mutagenesis, Mutants created were stably expresse d in AtT-SO cells. Regulated secretion was studied by analyzing basal and s timulated release of SST-14 LI and by immunocytochemistry for staining of S ST-14 LI in punctate granules. Deletion of the PSST-(3-15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unproce ssed precursor. Alanine scanning mutagenesis identified the region Pro(5)-G ln(12) as being important in precursor targeting, with Leu(7) and Leu(11) b eing critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha -helix. Di sruption of the alpha -helix did not impair the ability of PSST to be proce ssed at the COOH terminus to SST-14 and SST-28. Processing, however, was sh ifted to the early compartments of the secretory pathway rather than storag e granules and was relatively inefficient.