PROTEASOME AND THIOL INVOLVEMENT IN QUALITY-CONTROL OF GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR ADDITION

Improperly processed secretory proteins are degraded by a hydrolytic s ystem that is associated with the endoplasmic reticulum (ER) and appea rs to involve re-export of lumenal proteins into the cytoplasm for ult imate degradation by the proteasome. The chimaeric protein hGHDAF28, w hich contains a crippled glycosylphosphatidylinositol (GPI) C-terminal signal peptide, is degraded by a pathway highly similar to that for o ther ER-retained proteins and is characterized by formation of disulph ide-linked aggregates, failure to reach the Golgi complex and intracel lular degradation with a half life of similar to 2 h. Here we show tha t N-acetyl-leucinal-leucinal-norleucinal, MG-132 and lactacystin, all inhibitors of the proteasome, protect hGHDAF28; hGHDAF28 is still prot eolytically cleaved in the presence of lactacystin or MG-132, by the r emoval of similar to 2 kDa, but the truncated fragment is not processe d further. We demonstrate that the ubiquitination system accelerates E R-degradation of hGHDAF28, but is not essential to the process. Overal l, these findings indicate that GPI quality control is mediated by the cytoplasmic proteasome. We also show that the presence of a cysteine residue in the GPI signal of hGHDAF28 is required for retention and de gradation, as mutation of this residue to serine results in secretion of the fusion protein, implicating thiol-mediated retention as a mecha nism for quality control of some GPI signals. Removal of the cysteine also prevents inclusion of hGHDAF28 in disulphide-linked aggregates, i ndicating that aggregate formation is an additional retention mechanis m for this class of protein. Therefore our data suggest that an unpair ed terminal cysteine is the retention motif of the hGHDAF28 GPI-proces sing signal and that additional information may be required for effici ent engagement of ER quality control systems by the majority of GPI si gnals which lack cysteine residues.