Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro.

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.

Chromatin subunits contain normal levels of major acetylated histone species.

Chromatin subunits or nucleosomes prepared by micrococcal nuclease digestion of nuclei from trout testis have been examined for the presence of in vivo modified histone species. Both monomers and multimers are labeled when testis cells are incubated in the presence of [14C]acetate, but the level of radioactivity in the monomer fraction is 10 to 20% higher than in the multimer fraction. This difference is attributed to the trimming of nucleotides from the monomer DNA, associated with the loss of H1. The specific activities of the [14C]acetate label in histones H2A, H2B, H3, and H4 in monomer particles are similar to their respective values in whole chromatin. Starch gel electrophoresis of histone fractions derived from monomeric nucleosomes revealed the presence of monoacetylated and phosphorylated species of H2A, monoacetylated species of H2B and H3, and mono-, di-, tri-, and tetraacetylated species of H4. No differences in the content of these species between monomeric nucleosomes and whole chromatin could be discerned.

Histone methylation. Its occurrence in different cell types and relation to histone H4 metabolism in developing trout testis.

Histone methylation in developing trout testis has been observed in the diploid stem cells and primary spermatocytes, which actively synthesize DNA and histones. In spermatids, histone methylation is minimal and so probably plays no role in the replacement of histones by protamine which is characteristic of this cell type. No turnover of histone methyl groups could be detected over several hours, so that unlike acetylation or phosphorylation of histones, methylation in this tissue appears to be a stable, irreversible modification. When histone H4, labeled with [14C]methyl groups, is separated on starch gels into acetylated and phosphorylated derivatives, [14C]methyl label does not appear in positions characteristic of newly synthesized histone H4, i.e. the highly acetylated (di-, tri-, and tetra-acetylated), unphosphorylated species. [14C]Methyl label appears rather in the unphosphorylated, and unacetylated or monoacetylated species, shifting with time to the monophosphorylated form of histone H4. These data suggest a temporal sequence of events for histone H4: synthesis, then acetylation and deacetylation, followed by methylation and phosphorylation. Occurring late after histone synthesis and assembly into chromatin, histone methylation might then be necessary for histone interactions with other molecules (e.g. histone phosphokinase) prior to mitosis.