Characterization of rat100, a 300-kilodalton ubiquitin-protein ligase induced in germ cells of the rat testis and similar to the Drosophila hyperplastic discs gene.

Conjugation of ubiquitin to proteins is activated during spermatogenesis. Ubiquitination is mediated by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (UBCs or E2s), and ubiquitin protein ligases (E3s). Since we previously showed that the activated ubiquitination is UBC4 dependent, we characterized Rat100, a UBC4-dependent E3 expressed in the testis. Analysis of expressed sequence tag sequences and immunoblotting showed that Rat100 is actually a 300-kDa protein expressed mainly in the brain and testis and is similar to the human E3 identified by differential display (EDD) protein and the Drosophila hyperplastic discs gene, mutants of which cause a defect in spermatogenesis. Rat100 is induced during postnatal development of the rat testis, peaking at d 25. It is localized only in germ cells and is highly expressed in spermatocytes, moderately in round and slightly in elongating spermatids. In contrast to UBC4 whose removal from a testis extract abrogates much of the conjugation activity, immmunodepletion of Rat100 from the extracts had little effect. Rat100 therefore has a limited subset of substrates, some of which appear associated with the E3 as the immunoprecipitate containing Rat100 supported incorporation of (125)I-ubiquitin into high molecular weight proteins. Thus, Rat100 is the homolog of human EDD and likely of Drosophila hyperplastic discs. This homology, together with our results, suggests that induction of this E3 results in ubiquitination of specific substrates, some of which are important in male germ cell development.