Expression and assay of glycoprotein-specific ubiquitin ligases.

N-linked glycosylation of proteins that takes place in the endoplasmic reticulum (ER) plays a key role in protein quality control. Misfolded proteins or unassembled protein complexes that fail to achieve their functional states in the ER are retrotranslocated into the cytosol and degraded by the ubiquitin-proteasome system in a process called ER-associated degradation (ERAD). N-linked glycoprotein-specific ubiquitin ligase complexes, SCF(Fbs1) and SCF(Fbs2), appear to participate in ERAD for selective elimination of aberrant glycoproteins in the cytosol. This chapter describes methods employed for the isolation and oligosaccharide-binding assay of Fbs proteins that are the substrate-recognition components of the SCF(Fbs) complex and the in vitro ubiquitylation assay of the SCF(Fbs) ubiquitin ligase complexes.