Quaternary and Domain Structure of Glycoprotein Processing Glucosidase II

• Published in: Biochemistry (Easton) 2001-09, Vol.40 (35), p.10717-10722
• Main Authors: Trombetta, E. Sergio, Fleming, Karen G, Helenius, Ari
• Format: Article
• Language: English
• Subjects: alpha-Glucosidases - chemistry; alpha-Glucosidases - metabolism; Amino Acid Sequence; Animals; Cross-Linking Reagents; glucosidase II; Glycoproteins - metabolism; Molecular Sequence Data; Protein Processing, Post-Translational; Protein Structure, Quaternary; Protein Structure, Tertiary; Rats; Schizosaccharomyces pombe
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010629u

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.