This is, at least in part, mediated by BAG3 in association with the polyUb- and LC3-binding protein SQSTM1.
The hHR23A and B (100 nM) bound only to the polyUb-E6AP and not to polyUb-Ube2SΔ.
These two oriented helices in the Ub-bound form are likely beneficial to the binding with diUb, polyUb or ubiquitinated substrates, no matter whether K48- or K63-linked (Fig. 8C).
This also implies that polyUb chains that do not bind significantly with the 19S can still be targeted for disassembly by proteasome-associated DUBs.
On the other hand, some tandem motifs or domains, though non-cooperatively, can mediate formation of a complex or oligomer by binding with polyUb [47], [48].
Hydrolysis of substrates linked to homogenous non-forked Ub chains was not enhanced by addition of S5a and the authors therefore conclude that S5a interacts with the growing polyUb chain to prevent fork formation.
We find that homotypic K11-polyUb chains do not bind with sufficient affinity or avidity to the 26S to stimulate degradation of the protein substrate, and that there are clear differences in the affinity of K11- and K48-linked chains for the 26S.
The polyUb species were eluted with cation buffer B (50 mM ammonium acetate, 1 M NaCl, pH 4.5), exchanged into PBS buffer, pH 7.4, and concentrated to a final volume of 1 ml.
Further, shorter Lys chains may have different conformations compared with longer chains and this introduces an additional layer of regulation where the length of the polyUb chain may determine which UBD binds and thereby determining the outcome of ubiquitylation.
When resin-bound conjugates were incubated with HeLa cell extracts and the bound proteins visualized by SDS-PAGE, the Rad23 proteins and Rpn10 bound only the K48-polyUb chains.
