The Rate of Phaseolin Assembly Is Controlled by the Glucosylation State of Its N-Linked Oligosaccharide Chains.

Abstract

Many of the proteins that are translocated into the endoplasmic reticulum are glycosylated with the addition of a 14-saccharide core unit (Glc3Man9GlcNAc2) to specific asparagine residues of the nascent polypeptide. Glucose residues are then removed by endoplasmic reticulum-located glucosidases, with diglucosylated and monoglucosylated intermediates being formed. In this study, we used a cell-free system constituted of wheat germ extract and bean microsomes to examine the role of glucose trimming in the structural maturation of phaseolin, a trimeric glycoprotein that accumulates in the protein storage vacuoles of bean seeds. Removal of glucose residues from the N-linked chains of phaseolin was blocked by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin. If glucose trimming was not allowed to occur, the assembly of phaseolin was accelerated. Conversely, polypeptides bearing partially trimmed glycans were unable to form trimers. The effect of castanospermine on the rate of assembly was much more pronounced for phaseolin polypeptides that have two glycans but was also evident when a single glycan chain was present, indicating that glycan clustering can modulate the effect of glucose trimming on the rate of trimer formation. Therefore, the position of glycan chains and their accessibility to the action of glucosidases can be fundamental elements in the control of the structural maturation of plant glycoproteins.