Figure 4: The high conservation of the ubiquitin sequence permits FUBE affinity-enrichment of unanchored polyubiquitin from diverse species. (A) The ubiquitin sequence is highly conserved across Animalia, Fungi and Plantae kingdoms, demonstrated by the comparison of human (Homo sapiens), yeast (Saccharomyces cerevisiae) and plant (Arabidopsis thaliana) ubiquitin sequences (residues 1-76). Identical residues are denoted by asterisks (*), while highly similar amino acids are denoted by a colon (:). Note that only a single residue (P/S19) shows no similarity in amino acid properties between yeast and plants vs. human. The ubiquitin residues that contact the Znf-UBP domain of USP5 (our FUBE) are denoted by shading. Importantly, the Znf-UBP contact residues are 100% conserved across human, plants and yeast. (B) Unanchored polyubiquitin is captured from plant root tissue using the FUBE, revealing an increase of ubiquitin immunoreactivity (in house antibody), with increasing volumes of plant lysate and a fixed volume FUBE (0.5, 2.5, 5 and 10 mL of plant lysate). (C) The FUBE enriches more unanchored polyubiquitin chains from a ΔUBP14 (Δ) strain relative to an isogenic wild type UBP14 (WT) strain, as detected by anti-ubiquitin (VU-1) and anti-K48 polyubiquitin antibodies. Immunoblotting reveals unanchored K48-linked polyubiquitin species as long as Ub6 from the ΔUBP14 strain. A K48-immunoreactive band that does not co-migrate with commercial unanchored K48-linked polyubiquitin chains was also purified on the FUBE (denoted by an asterisk; *) that migrates just below the commercial K48 ubiquitin tetramer. (D) FUBE-purified yeast unanchored polyubiquitin was characterised by treatment with either the broad specificity USP2 catalytic core (USP2), or full length USP5 (the human homolog of yeast Ubp14). Only the band denoted by the asterisk was resistant to USP5 treatment (but not USP2), whilst FUBE-purified polyubiquitin that comigrates with commercial K48-linked standards was readily disassembled by both USP2 and USP5. (E) Unanchored polyubiquitin can be captured from an additional mammalian cell line, HEK293Ts (293T), using immobilised-FUBE (Znf-UBP domain of USP5). However, longer unanchored polyubiquitin (and less monoubiquitin) can be captured in pull-downs using a catalytically inactive mutant (C335A) of full-length USP5 (FL), which contains additional UBDs (at least two UBA domains). Control (Con) lane represents CNBr Seph 4B that has no protein immobilised. Purifications were performed essentially, as described for U20S and HeLa cells (Materials and Methods). In this case ~ 500 μg of HEK293T cell extract was captured on 30 μL each of Con-, FUBE- or FL-USP5- Sepharose and 100% of captured protein was loaded per lane. K48 and K63 lanes represent 0.25 μg of commercial standards and blot was detected using VU-1 anti-ubiquitin antibody. Numbers indicate ubiquitin moieties in the chains.