Fig. 4. The unmasked tail of Rb serves as an effective initiation region in vitro.

a Sketch of proteasome substrates consisting of the UBL domain of human HR23B with 10×His-tag at the N-terminus, followed by an E. coli DHFR domain and a tail (10×His-hUBL-DHFR-tail). b Degradation of 10×His-hUBL-DHFR-tail containing different C-terminal tails as indicated by purified mammalian 26S proteasomes. 100 μM MG132 was added to the reactions where indicated. The graph plots the relative amount of protein estimated by electronic autoradiography in SDS-PAGE gel bands (representative images are shown in Supplementary Fig. 3b) over time as a percentage of the initial protein amount from three repeat experiments. c Schematic representation of Flag-tagged human ODC protein. ODC contains an unstructured region at its C-terminal end. d Degradation of ODC and the indicated variants by purified mammalian 26S proteasomes as in b: Flag-tagged ODC (WT), Flag-tagged ODC in which the C-terminal tail (amino acids 425–461) is deleted (∆C (no tail)), Flag-tagged ODC in which amino acids 425–461 are replaced with amino acids 879-928 of Rb (∆C-Rb^879–928), and Flag-tagged ODC in which amino acids 425–461 are replaced with amino acids 761–810 of Rb (∆C-Rb^761–810). Representative gel images are shown in Supplementary Fig. 3d. The graph plots the relative band intensities from three repeat experiments. e Proposed model of E7-mediated destabilization of Rb. E7 induces Rb cleavage and also enhances its ubiquitination, both of which facilitate degradation of Rb by the proteasome. Exposure of the intrinsic disordered region of Rb by calpain cleavage markedly accelerates proteasomal degradation of Rb because the exposed region serves as an effective initiation region for proteasomal degradation. Source data are provided as a Source Data file.