Purification of poly-ubiquitinated proteins by S5a-affinity chromatography.
Poly-ubiquitination, the post-translational covalent conjugation of isopeptide-linked chains of ubiquitin to other target proteins, is the central signal for proteolytic degradation by the 26S proteasome complex. The S5a subunit of the 26S proteasome binds poly-ubiquitin chains containing four or more ubiquitins. We have used an immobilised glutathione-S-transferase (GST)-S5a fusion protein to purify poly-ubiquitinated proteins from mammalian tissues, with the intention of expanding the repertoire of known substrates of the ubiquitin pathway. A complex mixture of poly-ubiquitinated proteins was successfully purified from normal pig brain extract following induction of in vitro ubiquitination. Western blots of two-dimensional gels of this mixture showed at least two diagonal series of ubiquitin-positive spots. Individual spots in each series were separated by approximately 9 kDa suggesting that they represent poly-ubiquitinated proteins with increasing numbers of ubiquitins in the chains. S5a-binding proteins purified from ubiquitination-induced human placental extracts, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualised by Coomassie staining, contained a single major species with an apparent denatured molecular mass of approximately 60 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 S5a (Hiyama, H., et al., J Biol. Chem. 1999, 274, 28,019-28,025).
Layfield
R
R
School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. robert.layfield@nottingham.ac.uk
Tooth
D
D
Landon
M
M
Dawson
S
S
Mayer
J
J
Alban
A
A
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010920:Placenta / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D013552:Swine; D025801:Ubiquitin / Q000378:metabolism*
D011506:Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin