DYNAMICS OF UBIQUITIN CONJUGATION DURING ERYTHROID-DIFFERENTIATION IN-VITRO

To gain insight into the role of ubiquitin-mediated proteolysis in ery throid differentiation, levels of ubiquitin conjugating enzymes (E2s) and ubiquitin conjugates were analyzed during in vitro differentiation of murine erythroleukemic (MEL) cells, After 4 days of culture in the presence of the inducer dimethyl sulfoxide, MEL cells expressed high levels of the erythroid-specific proteins, globin, and band 3. During the same interval, cellular contents (mol/cell) of E2-14K, E2-25K, and E2-35K decreased up to similar to 5-fold; as suggested by results obt ained with E2-25K, this reflected a lower level of mRNA in differentia ting cells. Concentrations of these E2s changed more modestly during i n vitro differentiation, since cellular volume also decreased. Compari son of levels of the three E2s in undifferentiated MEL cells and retic ulocytes suggests that their concentrations remain fairly constant dur ing in vivo differentiation of proerythroblasts into reticulocytes. Th us, these components of the ubiquitin-mediated proteolytic pathway are likely to function constitutively during this interval. Two dimension al Western blots showed a broad spectrum of ubiquitin conjugates, incl uding free multiubiquitin chains, in undifferentiated MEL cells. As se en for several E2s, the concentration of ubiquitin conjugates (includi ng free chains) decreased modestly during in vitro differentiation. E2 -20K and E2-230K, which are abundant in reticulocytes, were low or abs ent in undifferentiated and differentiated MEL cells, In erythroid cel ls these two E2s are reticulocyte-specific; apparently MEL cells do no t differentiate far enough to allow induction of their expression.