HETEROGENEITY IN THE STRUCTURE OF THE UBIQUITIN CONJUGATES OF HUMAN ALPHA-GLOBIN

Only monoubiquitinated (and to a much lesser extent diubiquitinated) I -125-alpha globin is observed as a conjugated intermediate in the ATP- and ubiquitin (Ub)-dependent proteolysis of I-122-alpha globin cataly zed by an unfractionated reticulocyte lysate (Shaeffer, J. R. (1994) J . Biol. Chem. 269, 22205-22210). A monoubiquitinated with a dilute aci d reagent to selectively cleave the I-125-alpha globin moiety between residues 94 (Asp) and 95 (Pro). Analysis by sodium dodecyl sulfate-pol yacrylamide gel electrophoresis of the resulting polypeptides showed t hat the Ub(1)-alpha conjugate consisted of a mixture of molecules in w hich 57% had Ub attached to the amino-terminal two-thirds and 43% had Ub attached to the carboxyl-terminal one-third of the I-125-alpha glob in monomer. This amino- to carboxyl-terminal region ubiquitination rat io was essentially unchanged when a Ub(1)-alpha conjugate intermediate , pulse-labeled for 15 min in the presence of ubiquitin aldehyde to in hibit disassembly, was analyzed after (a) a 2-h chase incubation with excess nonradioactive alpha globin or (b) isolation and incubation sep arately as a proteolysis substrate. Similar analysis of the diubiquiti nated I-125-alpha globin (Ub(2)-alpha) conjugate molecules showed that , after pulse labeling, 58% were diubiquitinated within the amino-term inal two-thirds and 42% were monoubiquitinated within both amino- and carboxyl-terminal regions of the I-125-alpha globin moiety (a small am ount of molecules diubiquitinated within the carboxyl-terminal region may also be present) and that the relative amounts of these molecular types changed little, if any, during the chase incubation. This invari ance in the amino- to carboxyl-terminal region ubiquitination ratio du ring degradation of the Ub(1)-alpha and Ub(2)-alpha conjugates suggest s that I-125-alpha globin molecules ubiquitinated within either the am ino- or carboxyl-terminal regions were both intermediates in the prote olysis of the unconjugated substrate. This heterogeneous pattern of Ub conjugation may also occur during the proteolysis of other long-lived intracellular proteins.