QUALITY-CONTROL OF GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR ATTACHMENT IN MAMMALIAN-CELLS - A BIOCHEMICAL-STUDY

hGHDAF28 is a chimaeric protein consisting of human growth hormone fus ed to a crippled signal sequence for glycosylphosphatidylinositol (GPI )-anchor addition from decay-accelerating factor, and serves as a mode l for quality control of GPI-anchor addition. hGHDAF28 is retained in a pre-Golgi compartment and degraded intracellularly by a mechanism wi th similarity to that for other endoplasmic reticulum (ER)-retained pr oteins (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 concentr ations of dithiothreitol (DTT) accelerated loss of hGHDAF28 by degrada tion from cell lysates, without promoting secretion or alteration of d isulphide-bond distribution, in contrast to a number of other examples of ER-retained proteins where DTT alters disulphide-bond formation. A dditionally, degradation of hGHDAF28 was sensitive to pH, being promot ed at pH 6.0 and inhibited at pH 8.0; however, the latter effect was t ransient, indicating incomplete blockade. Degradation was also partial ly enhanced by depletion of ER calcium with thapsigargin, but this was again a partial and transient effect. Furthermore, degradation was te mperature sensitive, with a gradual decrease in rate observed at lower temperatures. However, a sharp decrease in turnover between 15 degree s C and 20 degrees C, indicative of a requirement for transport to a p ost-ER compartment, was not observed. Degradation of hGHDAF28 was inse nsitive to treatment with nocodozole or compounds preventing cytoplasm ic autophagy, suggesting that ER degradation is independent of classic al autophagy and microtubule-dependent processes. In addition, disrupt ion of N-glycosylation with tunicamycin, or inhibition of processing o f immature N-glycan chains with castanospermine or deoxynojirimycin, h ad little effect on the stability of hGHDAF28, suggesting that disrupt ion of the BiP/calnexin quality-control system by bulk cellular secret ory proteins does not influence the ER-degradation pathway of hGHDAF28 . Intermolecular hGHDAF28 cysteine bonds result in the formation of ag gregates which are probably important in the retention of the molecule . The insensitivity of this structure to reduction in vivo, together w ith the enhanced degradation rate, indicates that DTT mediates its eff ect on stability via a molecule involved in degradation of hGHDAF28, p ossibly a thiol-sensitive protease.