A conserved alpha-helix at the amino terminus of prosomatostatin serves as a 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 signal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH(2)-terminal segment of PSST consists of an amphipathic alpha-helix, which has been totally conserved throughout vertebrate evolution. We have analyzed the PSST-(3--15) region for sorting function by alanine scanning and deletional mutagenesis. Mutants created were stably expressed in AtT-20 cells. Regulated secretion was studied by analyzing basal and stimulated release of SST-14 LI and by immunocytochemistry for staining of SST-14 LI in punctate granules. Deletion of the PSST-(3--15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unprocessed precursor. Alanine scanning mutagenesis identified the region Pro(5)--Gln(12) as being important in precursor targeting, with Leu(7) and Leu(11) being critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha-helix. Disruption of the alpha-helix did not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, was shifted to the early compartments of the secretory pathway rather than storage granules and was relatively inefficient.
Mouchantaf
R
R
Fraser Laboratories, Department of Medicine, McGill University, Royal Victoria Hospital and Montreal Neurological Institute, Montreal, Quebec H3A 1A1, Canada.
Kumar
U
U
Sulea
T
T
Patel
Y C
YC
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D005455:Fluorescent Antibody Technique; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D013004:Somatostatin / Q000235:genetics* / Q000378:metabolism*
D011498:Protein Precursors; D013004:Somatostatin; C028456:prosomatostatin