Ubiquitin carrier protein-catalyzed ubiquitin transfer to histones. Mechanism and specificity.
Ubiquitinated derivatives of histones H2A and H2B, in which the carboxyl terminus of ubiquitin is joined to epsilon-amino groups of specific lysine residues of each histone, occur in vivo. Certain ubiquitin carrier proteins (E2s) catalyze ubiquitin transfer to histones (Pickart, C. M., and Rose, I. A. (1985) J. Biol. Chem. 260, 1573-1581). The catalytic activities of these purified ubiquitin carrier proteins have been quantitatively characterized with purified histones, in order to determine if one or more of them exhibits specificity for H2A over other histones (H3,H4) which are not known to be ubiquitinated in vivo. The results show the following. 1) No E2 exhibits strong specificity for H2A over the other histones. 2) For a given histone, kinetics of formation of its monoubiquitinated adduct do not differ strongly among the E2s; sigmoid kinetics (nH = 2) are generally observed, with values of K 0.5 ranging from 2-6 microM. 3) E214K catalyzes primarily monoubiquitination. 4) E220K catalyzes multiple ubiquitination (up to three ubiquitin/histone) by a processive mechanism that involves joining of ubiquitin carboxyl termini to multiple histone lysine residues. 5) E235K also catalyzes processive ubiquitination, with formation of polyubiquitinated products exhibiting a lag phase. Many of the polyubiquitinated adducts produced at low histone concentration are larger than expected for monoubiquitination of every histone-lysine residue, and polyubiquitination is selectively inhibited by substitution of reductively methylated ubiquitin for ubiquitin. These results suggest that E235K uniquely catalyzes ubiquitin transfer to lysine residues of previously conjugated ubiquitin molecule(s). The implications of these results for biological mechanisms of histone ubiquitination are discussed.
Pickart
C M
CM
Department of Biochemistry, State University of New York, Buffalo 14214.
Vella
A T
AT
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008025:Ligases; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D013950:Thymus Gland / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D002352:Carrier Proteins; D006657:Histones; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases