The E3 ubiquitin ligases HRD1 and SCF^Fbs2 recognize the protein moiety and sugar chains, respectively, of an ER-associated degradation substrate

Several E3 ubiquitin ligases have been identified that participate in endoplasmic reticulum-associated degradation (ERAD) of misfolded membrane and secretory proteins. Sometimes two ligases were shown to share the same substrate. However, it is not known whether they recognize the same structural determinants and constitute alternative pathways for ubiquitination or if they can act sequentially on different determinants on the substrate. Here we have analyzed in cells in vivo interactions of the recently identified mammalian HRD1 and SCF^Fbs2 with a model ERAD substrate, uncleaved precursor of asialoglycoprotein receptor H2a, and their involvement in its degradation. As Fbs2 had been shown to bind sugar chains, we studied the influence of glycosylation of the substrate by analyzing the effect of dominant negative mutants of HRD1 and Fbs2 on ERAD of H2a and of a mutant lacking all three of its glycosylation sites. Both HRD1 and Fbs2 were found to associate with H2a, and their mutants inhibited its degradation, but only dominant negative HRD1 blocked the degradation of the unglycosylated version of H2a. This suggests a cooperative action of the two ligases that target the glycans (Fbs2) and a protein motif (HRD1). In addition, our results suggest the need for retrotranslocation of the luminal glycosylated domain of the substrate prior to interaction with cytosolic Fbs2.