Quaternary and domain structure of glycoprotein processing glucosidase II

Glucose trimming from newly synthesized glycoproteins regulates their inter action with the calnexin/calreticulin chaperone system. We have recently pr oposed that glucosidase II consisted of two different subunits, alpha and b eta. The alpha subunit is the catalytic component, and deletion of its homo logue in yeast obliterates glucosidase II activity. Deletion of the homolog ue of the noncatalytic beta subunit in Schizosaccharomices pombe drasticall y reduces glucosidase II activity, but the role of the beta subunit in gluc osidase 11 activity has not been established. Furthermore, a direct interac tion between OL and fi subunits has not been demonstrated. Using chemical c ross-linking and hydrodynamic analysis by analytical ultracentrifugation, w e found that the two subunits form a defined complex, composed of one catal ytic subunit and one accessory subunit (alpha (1)beta (1)) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a high ly nonglobular shape. The asymmetric shape of the alpha (1)beta (1) complex was confirmed by its high susceptibility to proteases. The beta subunit co uld be proteolytically removed from the alpha (1)beta (1) complex without a ffecting catalysis, demonstrating that it is not required for glucosidase I I activity in vitro. Furthermore, we isolated a monomeric C-terminal fragme nt of the alpha subunit, which retained full glucosidase activity. We concl ude that the catalytic core of glucosidase 11 resides in a globular domain of the alpha subunit. which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodim eric complex with a highly extended conformation, which may favor interacti on with other proteins in the endoplasmic reticulum (ER). Through its C-ter minal HDEL signal, the beta subunit may retain the complete alpha (1)beta ( 1) complex in the ER.