Heterogeneity in the structure of the ubiquitin conjugates of human alpha globin

Only monoubiquitinated (and to a much lesser extent diubiquitinated) 125I-alpha globin is observed as a conjugated intermediate in the ATP- and ubiquitin (Ub)-dependent proteolysis of 125I-alpha globin catalyzed by an unfractionated reticulocyte lysate (Shaeffer, J.R. (1994) J. Biol. Chem. 269, 22205-22210). A monoubiquitinated 125I-alpha globin (Ub1-alpha) fraction was isolated and incubated with a dilute acid reagent to selectively cleave the 125I-alpha globin moiety between residues 94 (Asp) and 95 (Pro). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the resulting polypeptides showed that the Ub1-alpha conjugate consisted of a mixture of molecules in which 57% had Ub attached to the amino-terminal two-thirds and 43% had Ub attached to the carboxyl-terminal one-third of the 125I-alpha globin monomer. This amino- to carboxyl-terminal region ubiquitination ratio was essentially unchanged when a Ub1-alpha conjugate intermediate, pulse-labeled for 15 min in the presence of ubiquitin aldehyde to inhibit disassembly, was analyzed after (a) a 2-h chase incubation with excess nonradioactive alpha globin or (b) isolation and incubation separately as a proteolysis substrate. Similar analysis of the diubiquitinated 125I-alpha globin (Ub2-alpha) conjugate molecules showed that, after pulse labeling, 58% were diubiquitinated within the amino-terminal two-thirds and 42% were monoubiquitinated within both amino- and carboxyl-terminal regions of the 125I-alpha globin moiety (a small amount 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 invariance in the amino- to carboxyl-terminal region ubiquitination ratio during degradation of the Ub1-alpha and Ub2-alpha conjugates suggests that 125I-alpha globin molecules ubiquitinated within either the amino- or carboxyl-terminal regions were both intermediates in the proteolysis of the unconjugated substrate. This heterogeneous pattern of Ub conjugation may also occur during the proteolysis of other long-lived intracellular proteins.