Dynamics of ubiquitin conjugation during erythroid differentiation in vitro.
To gain insight into the role of ubiquitin-mediated proteolysis in erythroid 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 approximately 5-fold; as suggested by results obtained with E2-25K, this reflected a lower level of mRNA in differentiating cells. Concentrations of these E2s changed more modestly during in vitro differentiation, since cellular volume also decreased. Comparison of levels of the three E2s in undifferentiated MEL cells and reticulocytes suggests that their concentrations remain fairly constant during in vivo differentiation of proerythroblasts into reticulocytes. Thus, these components of the ubiquitin-mediated proteolytic pathway are likely to function constitutively during this interval. Two-dimensional Western blots showed a broad spectrum of ubiquitin conjugates, including free multiubiquitin chains, in undifferentiated MEL cells. As seen for several E2s, the concentration of ubiquitin conjugates (including free chains) decreased modestly during in vitro differentiation. E2-20K and E2-230K, which are abundant in reticulocytes, were low or absent in undifferentiated and differentiated MEL cells. In erythroid cells these two E2s are reticulocyte-specific; apparently MEL cells do not differentiate far enough to allow induction of their expression.
Haldeman
M T
MT
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214, USA.
Finley
D
D
Pickart
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology*; D008025:Ligases / Q000032:analysis / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases