Identification of N-glycan-binding proteins for E3 ubiquitin ligases.
N-glycans serve as a degradation signal by the SCF(Fbx2) ubiquitin ligase complex in the cytosol. Fbx2, an F-box protein, binds specifically to proteins attached with N-linked high-mannose type oligosaccharides, and subsequently contributes to ubiquitination of glycoproteins. Pre-integrin beta1 is identified as one of the Fbx2 targets. These two proteins bind in the cytosol after inhibition of the proteasome. These results indicate that SCF(Fbx2) ubiquitinates N-linked glycoproteins, which are translocated from the endoplasmic reticulum to the cytosol by the quality control mechanism. This chapter describes methods, including a binding protein assay for N-glycans, a ubiquitination assay for N-linked glycoproteins with SCF(Fbx2) ubiquitin ligase complex, an overlay assay for the detection of Fbx2 binding proteins, and a pull-down assay for the interaction between Fbx2 and N-linked glycoproteins, used to identify N-glycan-binding proteins for E3 ubiquitin ligases.
Tai
Tadashi
T
Seikagaku Corporation, Central Research Laboratories, Tokyo, Japan.
D000818:Animals; D001923:Brain Chemistry; D002460:Cell Line; D044783:F-Box Proteins / Q000378:metabolism; D006031:Glycosylation; D019012:Integrin beta1 / Q000378:metabolism; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D044783:F-Box Proteins; D019012:Integrin beta1; D011134:Polysaccharides; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases