FIGURE 1.

The UBA domain of SCCRO binds polyubiquitin chains in a linkage-independent manner. A, schematic depiction of the domain structure of SCCRO (top panel) and sequence analysis of the UBA domain of SCCRO with several other UBA and CUE domains (bottom panel). Highlighted are conserved hydrophobic residues critical for binding to ubiquitinated proteins. B, SCCRO preferentially binds Ub₄ over Ub or Ub₂. Lys⁴⁸-linked ubiquitin chains (Ub₂ and Ub₄) were synthesized in vitro. Pictured is a size exclusion chromatogram showing that SCCRO was co-eluted with Ub₄ but not with Ub or Ub₂. C, pulldown assay using GST-SCCRO and its mutants on Lys⁴⁸ or Lys⁶³ ubiquitin chains or HeLa cell lysates followed by Western blot analysis for ubiquitin showing that SCCRO binds to the ubiquitin chain in a linkage-independent manner. D, pulldown assay using GST-SCCRO on serially diluted Lys⁴⁸ or Lys⁶³ ubiquitin chains showing that SCCRO has preferential binding toward Lys⁶³-linked ubiquitin chains. E and F, immunoblot (IB) analysis of lysates from U2OS cells transfected with MYC-SCCRO and selected mutants probed with antibody against ubiquitinated proteins following immunoprecipitation (IP) for MYC showing that conserved hydrophobic residues in the UBA domain of SCCRO are critical for binding to ubiquitin chains. EV, empty vector.