Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains.
Radioiodinated histone H3 was incubated with ubiquitin, the ubiquitin-activating enzyme E1, and one of three ubiquitin carrier proteins, reticulocyte E2(20K) or E2(32K) or the yeast RAD6 product. Although the resulting ubiquitin-histone conjugates were synthesized in the absence of the substrate-binding protein E3, they were nevertheless degraded by purified rabbit reticulocyte 26 S protease. In contrast, unmodified histone H3 remained intact upon challenge with the 26 S ubiquitin/ATP-dependent enzyme. Conjugates produced by the RAD6 protein were better proteolytic substrates than those formed by reticulocyte E2 unless ubiquitin molecules with altered lysines were used for conjugate synthesis. Substitution of methylated ubiquitin or ubiquitin molecules in which lysine 48 was converted to arginine by site-directed mutation produced histone conjugates that were degraded at slow but measurable rates. Since methylated ubiquitin molecules are incapable of forming branched polyubiquitin chains, these results demonstrate that neither ubiquitin "trees" nor the substrate binding factor E3 is absolutely required for ubiquitin-dependent degradation of histone H3 in vitro.
Haas
A
A
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Reback
P M
PM
Pratt
G
G
Rechsteiner
M
M
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002474:Cell-Free System; D010450:Endopeptidases / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008025:Ligases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D044764:Ubiquitin-Activating Enzymes; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D002352:Carrier Proteins; D006657:Histones; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes