Specificity of Ubiquitin-Binding Proteins:Recognition of Different Faces of Ubiquitin

摘要

To further understand ubiquitin-binding proteins,we have developed a panel of monoubiquitin affinity resins linked through six different positions:residues 6,11,29,48,63,and 76.Each resin bound a different subset of yeast proteins.MALDI-TOF MS analysis of the eluted proteins identified several of these proteins.Adding excess free ubiquitin competes for the binding of specific ubiquitin-binding proteins.Thus,putative monoubiquitin-binding proteins could be identified by this method.Analysis of yeast protein with this panel of resins demonstrates clear differences in the protein-binding pattern,depending on what ubiquitin residue is coupled to the resin.The results suggested that certain proteins show a preference for binding to different faces of ubiquitin.Sapl85,an effector of the Sit4 protein phosphatase,has been identified as a putative ubiquitin-binding protein that binds to a face of ubiquitin other than that containing the hydrophobic patch.The combined affinity chromatography and mass spectrometry approach is a powerful tool for identifying ubiquitin-binding proteins.