DEGRADATION OF MONOUBIQUITINATED ALPHA-GLOBIN BY 26S PROTEASOMES

Ubiquitin-I-125-alpha-globin conjugate fractions containing either one (Ub(1)-alpha), two (Ub(2)-alpha), or a mixture of three and four (Ub( 3,4)-alpha) molecules of ubiquitin (Ub), covalently linked to one I-12 5-alpha-globin molecule were isolated after incubation of a proteolysi s reaction mixture containing ATP, ubiquitin aldehyde-treated reticulo cyte lysate, and human I-125-alpha-globin. Each of the purified conjug ate fractions or an identically-purified control sample of unconjugate d I-125-alpha-globin was incubated as a substrate in companion proteol ysis reaction mixtures containing either purified 26S or 20S rabbit re ticulocyte proteasomes. The initial rate of ATP-dependent degradation of the Ub(1)-alpha conjugate by the 26S proteasomes was similar to 0.4 4% (1.1 fmol)/min while that of the free I-125-alpha-globin was undete ctable. The initial rates of ATP-dependent degradation by the 26S prot easomes of the Ub(2)-alpha and Ub(3,4)-alpha conjugates conjugates wer e 2- to 3-fold that of the Ub(1)-alpha species. Conversely, the degrad ation of free I-125-alpha-globin and its ubiquitinated conjugates by t he 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 mixt ure with 26S proteasomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no conversion of the Ub(1)-alpha conjugate subs trate to higher-molecular-mass conjugates. These results suggest that monoubiquitinated alpha-globin can be degraded significantly and speci fically by interaction directly with the 26S proteasomes. This finding is consistent with the hypothesis that a substantial fraction of the Ub(1)-alpha conjugate intermediate in the ATP-dependent proteolysis of I-125-alpha-globin in whole reticulocyte lysate [Shaeffer, J. R. (199 4) J. Biol. Chem. 269, 22205-22210] is degraded by this interaction.