« Previous AbstractThe scope for using the volatile profiles of Pinus caribaea var. bahamensis as indicators of susceptibility to pine tortoise scale and as predictors of environmental stresses    Next Abstract"A synthetic analysis of the Saccharomyces cerevisiae stress sensor Mid2p, and identification of a Mid2p-interacting protein, Zeo1p, that modulates the PKC1-MPK1 cell integrity pathway" »

J Biol Chem

Title:		Secretion of somatostatin by Saccharomyces cerevisiae. Correct processing of an alpha-factor-somatostatin hybrid
Author(s):		Green R; Schaber MD; Shields D; Kramer R;
Address:
Journal Title:		J Biol Chem
Year:		1986
Volume:		261
Issue:		16
Page Number:		7558 - 7565
DOI:
ISSN/ISBN:		0021-9258 (Print) 0021-9258 (Linking)
Abstract:		"Somatostatin is a 14-amino acid peptide hormone that is proteolytically processed from its precursor, prosomatostatin, by a paired-basic-specific protease localized in the Golgi apparatus and secretory vesicles. Yeast (Saccharomyces cerevisiae MAT alpha) synthesize an analogous peptide hormone precursor, pro-alpha-factor, that contains tandem repeats of alpha factor (13 amino acids) flanked by spacers that include paired basic residues. To investigate the role of these two pro regions in mediating intracellular transport and processing, cloned genes specific for preprosomatostatin and prepro-alpha-factor were used to generate recombinants encoding hybrids between the alpha-factor pro region (amino-terminal) and somatostatin (carboxyl-terminal). These recombinants were inserted into yeast expression vectors under control of either the native alpha-factor promoter or the inducible yeast PHO5 (acid phosphatase) promoter. Yeast transformed with these plasmids expressed the hybrid messenger RNAs constitutively (alpha-factor promoter) or when induced in phosphate-deficient medium (PHO5 promoter). Radioimmunoassay of culture media revealed the secretion of up to 200 ng of immunoreactive somatostatin/10(7) cells. Metabolic labeling with [35S]cysteine, followed by immunoprecipitation with anti-somatostatin antibodies revealed two forms of hybrid precursor intracellularly, one of Mr 25,000, containing core carbohydrates, and a second of Mr 11,000, which was unglycosylated. Translation of mRNA extracted from these transformants in the wheat germ cell-free system revealed that the Mr 11,000 form was the primary translation product, whereas the Mr 25,000 species could be generated in vitro by inclusion of mammalian rough microsomes. The secreted immunoreactive material was shown to be authentic somatostatin by high pressure liquid chromatography analysis and protein sequencing. These results demonstrate that the yeast processing enzymes recognize these chimeric precursors, resulting in the secretion of the mature peptide hormone"
Keywords:		"Chromatography, High Pressure Liquid DNA, Recombinant/*metabolism Endoplasmic Reticulum/analysis Mating Factor Molecular Weight *Peptide Biosynthesis Peptides/genetics Protein Precursors/analysis/metabolism Saccharomyces cerevisiae/genetics/*metabolism So;"
Notes:		"MedlineGreen, R Schaber, M D Shields, D Kramer, R eng 5T32CA09060/CA/NCI NIH HHS/ AM-21860/AM/NIADDK NIH HHS/ AM01208/AM/NIADDK NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1986/06/05 J Biol Chem. 1986 Jun 5; 261(16):7558-65"