Purification of poly-ubiquitinated proteins by S5a-affinity chromatography

Poly-ubiquitination, the post-translational covalent conjugation of isopept ide-linked chains of ubiquitin to other target proteins, is the central sig nal for proteolytic degradation by the 265 proteasome complex. The S5a subu nit of the 26S proteasome binds poly-ubiquitin chains containing four or mo re ubiquitins. We have used an immobilised glutathione-S-transferase (GST)- S5a fusion protein to purify poly-ubiquitinated proteins from mammalian tis sues, with the intention of expanding the repertoire of known substrates of the ubiquitin pathway. A complex mixture of poly-ubiquitinated proteins wa s successfully purified from normal pig brain extract following induction o f in vitro ubiquitination. Western blots of two-dimensional gels of this mi xture showed at least two diagonal series of ubiquitin-positive spots. Indi vidual spots in each series were separated by approximately 9 kDa suggestin g that they represent poly-ubiquitinated proteins with increasing numbers o f ubiquitins in the chains. S5a-binding proteins purified from ubiquitinati on-induced human placental extracts, resolved by sodium dodecyl sulfate pol yacrylamide gel electrophoresis and visualised by Coomassie staining, conta ined a single major species with an apparent denatured molecular mass of ap proximately SO kDa. Edman degradation identified this protein as hHR23B, a human homologue of the Saccharomyces cerevisiae DNA repair protein Rad23p. In this case hHR23B is not ubiquitinated but instead contains an intrinsic ubiquitin-like domain at its N-terminus, through which it interacts with S5 a.