Figure 7
From: Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex
In vitro ubiquitination of recombinant β-catenin, MyHCemb and Alix mediated by CRL5Ozz. (a,d) GST-tagged-β-catenin, (b,e) MyHCemb fragment (1041–1941 a.a.) and (c,f) Alix was incubated with CRL5Ozz and either native ubiquitin or mutant Ub (K48R). In the presence of native ubiquitin and CRL5Ozz, β-catenin or MyHCemb or Alix were efficiently ubiquitinated. If the assay was performed in the presence of Ub K48R, the ubiquitination was reduced to background levels, demonstrating that CRL5Ozz polyubiquitinated β-catenin, MyHCemb and Alix. (a–c) CRL5Ozz efficiently ubiquitinated β-catenin, MyHCemb and Alix (lane 1). The specificity of the reaction was confirmed by omitting either the substrate (lane 2) or the CRL5Ozz complex (lane 3) from the ubiquitination assay, which significantly reduced the Ub-substrate products. (d–f) In the presence of native ubiquitin, CRL5Ozz efficiently ubiquitinated its substrates (lane 1). Instead, by using K48R Ub mutant in the ubiquitination reaction, the ubiquitination of the substrates was significantly reduced (lane 2).
