Levels of active ubiquitin carrier proteins decline during erythroid maturation.
Energy-dependent proteolysis is lost during maturation of rabbit reticulocytes to erythrocytes (Speiser, S., and Etlinger, J.D. (1982) J. Biol Chem. 257, 14122-14127), but nothing is known about the fates of individual components in the multienzyme ATP- and ubiquitin (Ub)-dependent proteolytic pathway during this process. Rabbit reticulocytes contain five low molecular weight carrier proteins (E2s) that form labile Ub adducts in the presence of Ub-activating enzyme (E1) (Pickart, C. M. and Rose, I. A. (1985) J. Biol. Chem. 260, 1573-1581). A method to estimate levels of active E2s in erythroid cells has been developed involving: 1) stepwise anion exchange fractionation of a soluble lysate; 2) addition of purified E1, MgATP, and radioiodinated Ub to the fractions followed by gel electrophoresis of the resulting E2-Ub adducts; and 3) quantitative densitometry of autoradiographs. Levels of active E2s are much lower in (rabbit) erythrocytes than in reticulocytes. Mean -fold decreases are: E235K, 6 x; E2(25K), 11 x; E2(20K), 18 x; E2(17K), not detected in erythrocytes; E2(14K), 12 x. The large decreases in levels of E2(20K) and E2(14K) are consistent with known functions of these proteins in DNA repair and Ub-dependent proteolysis, respectively. Decreases in levels of the other E2s, whose biological roles are presently unknown, suggest diminished requirements, if any, for them in erythrocyte metabolism. The analysis revealed two previously undescribed carrier proteins, one of which has a high molecular weight. Additional catalytic properties of E2(35K) and E2(14K) are reported.
Pickart
C M
CM
Department of Biochemistry, State University of New York, Buffalo 14214.
Vella
A T
AT
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D002352:Carrier Proteins / Q000097:blood*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004905:Erythrocyte Aging; D004912:Erythrocytes / Q000032:analysis / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008025:Ligases / Q000097:blood; D008970:Molecular Weight; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000097:blood
D002352:Carrier Proteins; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases