Figure 1. Synthesis and processing of ubiquitin precursors.

(a) Schematic representation of the four mammalian ubiquitin precursors. UBA52 and UBA80 are ubiquitin-ribosomal protein fusions (Ub-RPs) in which a single ubiquitin molecule is linked to a ribosomal protein, L40 and S27A, respectively. UBB and UBC are ubiquitin polymers (polyUbs) of different lengths followed by a C-terminal extension (ext) that ranges from a single amino acid (e.g., a cysteine and a tyrosine in human and mouse UBB, respectively) to a small polypeptide (e.g., 50 amino acid residues in mouse UBC). The number of ubiquitins in mouse UBB and UBC is 4 and 9; human UBB and UBC have 3 and 9 ubiquitins, respectively. Generation of free ubiquitin from these precursors requires the action of deubiquitinases (DUB; dashed arrows). (b) ³⁵S-labeled mouse Ub-RPs UBA52 and UBA80 and (c) mouse polyUbs UBB and UBC were synthesized in vitro in the absence or presence of DUB inhibitors (HA-UbVME or HA-Ubal), as indicated. Samples were withdrawn at the indicated time points. UBA52, UBA80, its corresponding processed products (L40, S27A and Ub), and partially processed UBB and UBC species are indicated. Note that S27A co-migrates with the abundant hemoglobin from the reticulocyte lysate and therefore is barely visible at earlier time points. In (b,c) numbers to the left indicate the molecular weights of protein standards in kDa.