title: Quality control of glycosylphosphatidylinositol anchor attachment in mammalian cells: a biochemical study.
abstract: hGHDAF28 is a chimaeric protein consisting of human growth hormone fused to a crippled signal sequence for glycosylphosphatidylinositol (GPI)-anchor addition from decay-accelerating factor, and serves as a model for quality control of GPI-anchor addition. hGHDAF28 is retained in a pre-Golgi compartment and degraded intracellularly by a mechanism with similarity to that for other endoplasmic reticulum (ER)-retained proteins (Field, Moran, Lee, Keller and Caras (1994) J. Biol. Chem. 269, 10830-10837). We have studied the specific pathway of degradation for hGHDAF28 using a number of compounds which affect protein folding and trafficking pathways in eukaryotic cells. We found that high concentrations of dithiothreitol (DTT) accelerated loss of hGHDAF28 by degradation from cell lysates, without promoting secretion or alteration of disulphide-bond distribution, in contrast to a number of other examples of ER-retained proteins where DTT alters disulphide-bond formation. Additionally, degradation of hGHDAF28 was sensitive to pH, being promoted at pH 6.0 and inhibited at pH 8.0; however, the latter effect was transient, indicating incomplete blockade. Degradation was also partially enhanced by depletion of ER calcium with thapsigargin, but this was again a partial and transient effect. Furthermore, degradation was temperature sensitive, with a gradual decrease in rate observed at lower temperatures. However, a sharp decrease in turnover between 15 degrees C and 20 degrees C, indicative of a requirement for transport to a post-ER compartment, was not observed. Degradation of hGHDAF28 was insensitive to treatment with nocodozole or compounds preventing cytoplasmic autophagy, suggesting that ER degradation is independent of classical autophagy and microtubule-dependent processes. In addition, disruption of N-glycosylation with tunicamycin, or inhibition of processing of immature N-glycan chains with castanospermine or deoxynojirimycin, had little effect on the stability of hGHDAF28, suggesting that disruption of the BiP/calnexin quality-control system by bulk cellular secretory proteins does not influence the ER-degradation pathway of hGHDAF28. Intermolecular hGHDAF28 cysteine bonds result in the formation of aggregates which are probably important in the retention of the molecule. The insensitivity of this structure to reduction in vivo, together with the enhanced degradation rate, indicates that DTT mediates its effect on stability via a molecule involved in degradation of hGHDAF28, possibly a thiol-sensitive protease.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, U.K.
authors.forename: L J
authors.initials: LJ
authors.lastname: Wainwright
authors.forename: M C
authors.initials: MC
authors.lastname: Field
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000235:genetics; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013696:Temperature; D014162:Transfection / Q000235:genetics
chemical_list: D017261:Glycosylphosphatidylinositols; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D002118:Calcium; D004229:Dithiothreitol