Functional heterogeneity of ubiquitin carrier proteins.
In the formation of covalent ubiquitin-protein conjugates that occurs during ATP- and ubiquitin-dependent proteolysis in reticulocyte extracts, ubiquitin (Ub) is activated to a thiol ester of the activating enzyme E1 (via the Ub carboxyl terminus), transferred to low-molecular weight "carrier proteins" (E2s) to form E2-Ub thiol esters, and then transferred by a third enzyme (E3) to amino groups on target proteins (Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206-8214). We report here the fractionation of Ub carrier proteins by molecular weight, and their characterization with respect to several activities. The Ub thiol ester forms of at least four of the five E2s catalyze Ub transfer to a number of small amines, in a reaction that does not require E3; only primary amines on primary carbons can serve as Ub acceptors. E3-independent Ub transfer to the small, basic proteins histones H2A and H2B, and cytochrome c, is also observed. The Ub thiol ester forms of two of the E2s were found to catalyze Ub transfer to cytochrome c. Only a single E2 functions in E3-dependent conjugate formation (with the substrates creatine phosphokinase, reduced/carboxymethylated serum albumin, and oxidized RNase) and in E3-dependent protein breakdown (with the substrate serum albumin). This E2 has a subunit molecular weight of 14,000 and migrates as a dimer on Sephacryl 200.
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D000588:Amines / Q000378:metabolism; D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism; D004952:Esters; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D000588:Amines; D003574:Cytochrome c Group; D004952:Esters; D006609:High Mobility Group Proteins; D006657:Histones; D010710:Phosphates; D013438:Sulfhydryl Compounds; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D008239:Lysine