Fig. 1: Chemical synthesis and ubiquitination of ubiquitin-conjugates.
From: The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag
a A cartoon depicting cyclin B1 with its 1–88 aa residue unstructured N-terminal region (NT), and a C-Terminal CDK1 binding region (left). The primary amino acid sequence of the N-terminal region of cyclin B1 that encompasses the Degron and 15 Lysine residues (right). Below are the wild-type (WT) and K0-mutant (all Lysine to Arginine) Cyclin B1-NT sequences used in this study for mammalian cell expression. The K64 HA-Cyclin B1-NT sequence was used for chemical ubiquitination of substrates (bottom). b Schematic illustration of the strategy used to synthesize differentially tagged TetraUbK48-HA-Cyclin B1-NT (Nbz = N-acyl-benzimidazolinones, SR = 3-mercaptopropionic acid). The detailed chemical synthesis protocol is described in Supplementary Methods. c A cartoon representation of a set of four synthetic substrates designed to study proteasome function. Tagged (Myc/Flag) and/or untagged ubiquitin units were attached to lysine64 of the HA-Cyclin B1-NT to obtain ubiquitinated conjugates. The numbers 3, 5, and 7 correspond to the three synthetic substrates in panel (b) (see also Supplementary Methods). d All four synthetic ubiquitinated Cyclin B1-NT conjugates were resolved by SDS-PAGE and stained with Coomassie or probed with the indicated antibodies for immunoblotting (IB). e Human 30S/26S and 20S proteasomes purified from human erythrocytes were resolved by native gel for in-gel activity (left), or for Coomassie (center), or resolved by SDS-PAGE for Coomassie staining. The band marked by * near 90 kDa was identified as HSP90AA1 and HSP90AB1 in MS/MS analysis. All other bands were confirmed to contain primarily 20S (right) or 26S (to its left) subunits. Source data are provided as a Source data file.
