Ubiquitinated Histones and Chromatin

As the first enzyme in the ubiquitin system the ubiquitin-activating enzyme El plays a pivotal role in all pathways of protein ubiquitination. In an effort to learn more about the cell biology of this pathway, we have purified the llO-kDa enzyme to homogeneity and generated a panel of distinct monoclonal antibodies to it. Using quantitative electron microscopic immunolocalization with these anti-El monoclonal antibodies, we rind that El is abundant both within the cytoplasm and nucleus. Within the cytoplasm, El was found throughout the cytoplasmic volume as well as enriched along the cytoplasmic face of the rough endoplasmic reticulum and associated with the dense material along the desmosomal junctions. El was also found associated with the cytoplasmic surface of endosomal/lysosomal vacuoles. Interestingly, El was also found within the mitochondria. The lumen of rough endoplasmic reticulum, Golgi complex, endosomes, and lysosomes were negative. The specific localization of El to distinct subceilular organelles suggests that El may play multiple physiological roles within the cell.

mentioning

confidence: 99%

Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.

Schwartz

Trausch

Ciechanover

et al. 1992

“…Stable ubiquitin acceptors include histones H2A and H2B (4), actin (5), the growth hormone receptor (6), and the lymphocyte homing receptor (7). The existence of these stable ubiquitin-protein conjugates is apparently explained by the observation that these proteins are monoubiquitinated and therefore do not activate the ubiquitin-dependent protease.…”

mentioning

confidence: 99%

Identification of a viral gene encoding a ubiquitin-like protein.

Guarino

1990

“…Evidence suggests that ubiquitin conjugation may have other roles in the cell as well. Two of the most abundant ubiquitin conjugates in mammalian cells are adducts formed with histones H2A and H2B (17). Because the ubiquitin moiety of H2A and H2B conjugates is in rapid equilibrium with the cellular pool of free ubiquitin (18) and because histones have such long half-lives, these adducts have generally not been considered intermediates of degradation, although this possibility has not been ruled out.…”

mentioning

confidence: 99%

“…Because the ubiquitin moiety of H2A and H2B conjugates is in rapid equilibrium with the cellular pool of free ubiquitin (18) and because histones have such long half-lives, these adducts have generally not been considered intermediates of degradation, although this possibility has not been ruled out. The levels of ubiquitin-histone conjugates have been observed to fluctuate with respect to the cell cycle (17,19) and transcriptional activity (20). More recently, two genes in the yeast Saccharomyces cerevisae, RAD6 (4) and CDC34 (21), were discovered to be E2 proteins capable of in vitro, E3-independent ligation of ubiquitin to histones.…”

mentioning

confidence: 99%

A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6.

Sullivan

Vierstra

1989

The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation. Here we identify an E2 from a higher plant, wheat, that is similar toRAD6 with respect to both structure and in vitro substrate specificity. The Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic. Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants.Ubiquitin has several roles in eukaryotic cells as a result of its covalent ligation to various intracellular proteins (1, 2). Ubiquitin ligation requires ATP and is catalyzed by a multienzyme pathway initially described in rabbit reticulocyte lysates (3) and subsequently characterized in yeast (4) and wheat germ extracts (5). In the initial step of ligation, ubiquitin-activating enzyme (El) adenylates the carboxyl terminus of ubiquitin by using ATP. The activated molecule is then attached by means of a thiol ester linkage to a thiol site on El with the concomitant release of AMP (6). The ubiquitin moiety is next transferred to a reactive cysteine on ubiquitin carrier proteins (E2) by transesterification. The E2 proteins comprise a family of low molecular weight proteins that differ in their recognition of specific target proteins and in their requirement for a ubiquitin protein ligase (E3) (7-11). Finally, ubiquitin is covalently attached to target proteins with or without the participation of E3. The bond thus formed is an isopeptide linkage between the carboxyl terminus of ubiquitin and iysyl e-amino groups within the target protein.The best characterized of ubiquitin's functions is its role in selective proteolysis, where conjugation serves to commit proteins to degradation (2, 12). Both in vitro and in vivo studies have provided evidence for a causal relationship between the formation of specific ubiquitin-protein conjugates and the degradation of target proteins (13-16). Evidence suggests that ubiquitin conjugation may have other roles in the cell as well. Two of the most abundant ubiquitin conjugates in mammalian cells are adducts formed with histones H2A and H2B (17). Because the ubiquitin moiety of H2A and H2B conjugates is in rapid equilibrium with the cellular pool of free ubiquitin (18) and because histones have such long half-lives, these adducts have generally not been considered intermediates of degradation, although this possibility has not been ruled out. The levels of ubiquitin-histone conjugates have been observed to fluctuate with respect to the cell cycle (17, 19) and transcriptional activity (20). More recently, two genes in the yeast Saccharomyces cerevisae, RAD6 (4) and CDC34 (21), were discovered to be E2 proteins capable of in vitro, E3-independent ligation of ubiquitin to histones. The RAD6 gene has been implicated in DN...