Quaternary and Domain Structure of Glycoprotein Processing Glucosidase II

Abstract

Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, α and β. The α subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic β subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the β subunit in glucosidase II activity has not been established. Furthermore, a direct interaction between α and β subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (α₁β₁) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the α₁β₁ complex was confirmed by its high susceptibility to proteases. The β subunit could be proteolytically removed from the α₁β₁ complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the α subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase II resides in a globular domain of the α subunit, which can function independently of the β subunit, while the complete α and β subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the β subunit may retain the complete α₁β₁ complex in the ER.