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.
Mesh Terms:
Animals, Antigens, CD29, Brain Chemistry, Cell Line, F-Box Proteins, Glycosylation, Mice, Polysaccharides, SKP Cullin F-Box Protein Ligases, Ubiquitin-Protein Ligases
Meth. Enzymol.
Date: Nov. 23, 2006
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