title: Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells.
abstract: Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that approximately 68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of approximately 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.
authors.affiliation: Brain Disease Research Center, School of Medicine, Ajou University, Suwon 442-721, Korea.
authors.forename: Myung Ae
authors.initials: MA
authors.lastname: Lee
authors.forename: Kwang Ho
authors.initials: KH
authors.lastname: Cheong
authors.forename: Dennis
authors.initials: D
authors.lastname: Shields
authors.forename: Sang Dai
authors.initials: SD
authors.lastname: Park
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Hong
mesh_terms: D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000166:cytology*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism; D013004:Somatostatin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013004:Somatostatin; D000072235:Mating Factor