An uncleaved glycosylphosphatidylinositol signal mediates Ca(2+)-sensitive protein degradation.
Inv-gp80 is a chimeric protein which contains a signal for the attachment of a glycosylphosphatidylinositol (GPI) anchor. When expressed in Dictyostelium discoideum, this protein fails to become GPI anchored and is retained within the cell as an integral membrane protein. We have compared the subcellular localization and degradation of Inv-gp80 with that of its intracellular but soluble counterpart, Inv-gp80sc. Inv-gp80sc lacks the hydrophobic C-terminal 22 amino acids of Inv-gp80. The N-linked oligosaccharides of both Inv-gp80 and Inv-gp80sc remained sensitive to endoglycosidase H, and both proteins co-fractionated with endoplasmic reticulum marker enzymes on Percoll gradients. Under normal conditions, Inv-gp80 displayed a half-life (t 1/2) of 90 min, while Inv-gp80sc displayed a t 1/2 of 120 min. The degradation of both proteins required ATP, was inhibited by tosyl phenylalanylchloromethane (Tos-Phe-CH2Cl) and was insensitive to inhibitors of lysosomal function. While depletion of Ca2+ from the endoplasmic reticulum had no effect on the degradation of Inv-gp80sc, it stimulated the degradation of Inv-gp80. When the GPI anchor signal sequence of Inv-gp80 was replaced with the transmembrane domain of the interleukin-2 receptor, the degradation of the protein was no longer influenced by Ca2+ fluxes. The data suggest that while the GPI anchor sequence of Inv-gp80 does not contain determinants regulating the degradation of the protein under basal conditions, it targets Inv-gp80 for rapid degradation under conditions where Ca2+ is depleted from the endoplasmic reticulum.
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E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, MO 63104, USA.
Klein
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D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D004023:Dictyostelium / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006207:Half-Life; D006596:Hexosaminidases / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012995:Solubility; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D043324:beta-Fructofuranosidase
D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D007476:Ionophores; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C052291:cell cohesion molecule, Dictyostelium; D000001:Calcimycin; D014108:Tosylphenylalanyl Chloromethyl Ketone; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D043324:beta-Fructofuranosidase; D002118:Calcium