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Departments of Medicine (R.O., N.B., O.A.J.A., V.R., S.S.W.), of Anatomy and Cell Biology (C.R.M.), and of Pediatrics, Pharmacology and Human Genetics, and Montréal Children’s Hospital Research Institute (J.T.), McGill University, Montréal, Québec, Canada H3A 2B2
Address all correspondence and requests for reprints to: S. S. Wing, Polypeptide Laboratory, McGill University, Strathcona Anatomy and Dentistry Building, 3640 University Street, Room W315, Montréal, Québec, Canada H3A 2B2. E-mail: simon.wing{at}mcgill.ca.
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 125I-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.
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