Pre-Golgi degradation of yeast prepro-alpha-factor expressed in a mammalian cell. Influence of cell type-specific oligosaccharide processing on intracellular fate.
We demonstrate that expression of yeast prepro-alpha-factor in GH3 rat pituitary cells results in its degradation in an endoplasmic reticulum (ER) or early Golgi compartment, suggesting that this wild-type prohormone is recognized as abnormal or misfolded in the context of a mammalian ER. In GH3 cells, as in yeast, the nascent polypeptide is efficiently targeted to the ER, where it undergoes cleavage of its amino-terminal signal peptide and core glycosylation to form glycosylated pro-alpha-factor (gp alpha f). Subsequently, however, this species disappears from cells with a half-time of 25-30 min (including a 10-20-min lag), and no alpha-factor- or proregion-derived products can be detected. Localization of the degradative process to the ER is suggested by its occurrence in the presence of brefeldin A or chloroquine and by the endoglycosidase H susceptibility of the substrate. We present evidence that Asn-linked oligosaccharide processing, which differs in extent between yeast and mammalian cells, may be an important factor in determining degradation in this heterologous circumstance. When GH3 cells are treated with deoxymannojirimycin, an inhibitor of ER alpha-mannosidases, gp alpha f is essentially stable, suggesting that trimming of core oligosaccharides below Man8 (a process that does not occur in yeast) strongly promotes proteolysis. Inhibition of ER glucosidase activity by treatment with deoxynojirimycin, by contrast, considerably accelerates the disappearance of gp alpha f (t1/2 = 8-10 min). These data indicate that cell type-specific post-translational modifications of a secretory glycoprotein can substantially modify its recognition by the mammalian ER degradative apparatus.
Su
K
K
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029.
Stoller
T
T
Rocco
J
J
Zemsky
J
J
Green
R
R
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D020126:Brefeldin A; D002240:Carbohydrate Sequence; D003517:Cyclopentanes / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021382:Protein Sorting Signals / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D003517:Cyclopentanes; D005656:Fungal Proteins; C048632:MF(ALPHA)1 protein, S cerevisiae; D009844:Oligosaccharides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014415:Tunicamycin; D017485:1-Deoxynojirimycin; D020126:Brefeldin A