Degradation of monoubiquitinated alpha-globin by 26S proteasomes.
Ubiquitin-125I-alpha-globin conjugate fractions containing either one (Ub1-alpha), or two (Ub2-alpha), or a mixture of three and four (Ub3,4-alpha) molecules of ubiquitin (Ub), covalently linked to one 125I-alpha-globin molecule were isolated after incubation of a proteolysis reaction mixture containing ATP, ubiquitin aldehyde-treated reticulocyte lysate, and human 125I-alpha-globin. Each of the purified conjugate fractions or an identically-purified control sample of unconjugated 125I-alpha-globin was incubated as a substrate in companion proteolysis reaction mixtures containing either purified 26S or 20S rabbit reticulocyte proteasomes. The initial rate of ATP-dependent degradation of the Ub1-alpha conjugate by the 26S proteasomes was approximately 0.44% (1.1 fmol)/min while that of the free 125I-alpha-globin was undetectable. The initial rates of ATP-dependent degradation by the 26S proteasomes of the Ub2-alpha and Ub3,4-alpha conjugates were 2- to-3-fold that of the Ub1-alpha species. Conversely, the degradation of free 125I-alpha-globin and its ubiquitinated conjugates by the 20S proteasomes was not dependent on ATP, nor did it increase with the size of the Ub adduct. Analysis of the products of a reaction mixture with 26S proteasomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no conversion of the Ub1-alpha conjugate substrate to higher-molecular-mass conjugates. These results suggest that monobiquitinated alpha-globin can be degraded significantly and specifically by interaction directly with the 26S proteasomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Shaeffer
J R
JR
Center for Blood Research, Brigham and Women's Hospital, Boston, Massachusetts.
Kania
M A
MA
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005914:Globins / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D014452:Ubiquitins / Q000378:metabolism*
D007457:Iodine Radioisotopes; D014452:Ubiquitins; D005914:Globins; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease