Abstract
Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.
Highlights
Ns (GNs 194 and 197–199), it did bind a single isolated sulfated protein (GN 26) (Fig. 2D)
Summary of the diverse properties reported for the FBA family in this study Binding of FBA proteins to high containing glycoproteins was determined by ConA blotting of proteins co-immunoprecipitated with FBA family members
Our results reveal marked functional differences among the FBA proteins, beginning with differing affinity for high mannose-containing glycoproteins
Summary
Gene column lists the HUGO designation of the gene and reference GenBankTM accession numbers. Location refers to nucleotide in reference gene or, in the case of the Null mutants, the location from the FBXO start site. Expected products indicate length of gene cloned or, in the case of the Null mutants, the size of the entire plasmid obtained using QuikChange mutagenesis.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have