Exp Mol Med.  2002 Sep;34(4):285-293.

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells

• Lee MA
• Cheong KH
• Shields D
• Park SD
• Hong SH

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 -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 - 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.

Keyword

yeast alpha-factor; peptide hormone processing; heterologous expression; GH3 cell; secretory pathway

MeSH Terms

Animals
Cell Line
Endoplasmic Reticulum/metabolism
Golgi Apparatus/metabolism
Kinetics
Peptides/genetics/*metabolism
Pituitary Gland, Anterior/*cytology
Protein Precursors/biosynthesis/genetics/*metabolism
*Protein Processing, Post-Translational
Protein Sorting Signals/genetics
Protein Transport
Rats
Recombinant Proteins/biosynthesis/metabolism
Retroviridae/genetics
Saccharomyces cerevisiae/genetics/*metabolism
Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism
Secretory Vesicles/metabolism
Somatostatin/biosynthesis/genetics/metabolism/secretion