Studies on cellular basis of the endoplasmic reticulum-associated degradation and intracellular aggregation of misfolded proteins

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The endoplasmic reticuium (ER) is known to function as a quality control machinery of nascent proteins, and various misfolded or unassembled proteins in the ER are targeted to ubiquitin/proteasome-mediated degradation after retrotranslocation to the cytosol through the Sec61 translocon. We have shown that not only proteasome inhibitors such as lactacystin and carbobenzoxy-leucyl-leucyl-leucinal (LLL), but also inhibitors of ER mannosidase I such as kifunensine and deoxyrnannojirimycin, strongly inhibit the ERAD of various misfolded proteins, suggesting that processing of N-linked oligosaccharide plays an important role on ERAD. In this study, collaborating with Dr. Yoshida in Tokyo Metropolitan Institute of Medical Science, we found that N-glycan serves as a signal for degradation by the Skpl-Cullin1-Fbx2-Rbx1 (SCF^<Fbx2>) ubiquitin ligase complex. The F-box protein Fbx2 binds specifically to proteins attached to N-inked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. In addition, expression of the mutant Fbx2ΔF, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ERAD pathway. Our results indicate that SCF^<Fbx2> ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.