Abstract
In Drosophila melanogaster, the dual E3 Ubiquitin/SUMO-1 ligase dTopors localizes to the spermatocyte nuclear lamina and is required for nuclear morphology and meiotic chromosome transmission. To investigate the role of dTopors-mediated ubiquitination, we use CRISPR/Cas9 to mutate the ubiquitination domain. Two mutants were recovered: C105A, a substitution of a conserved cysteine, and Df(114-118), a hypomorphic mutant with an altered ubiquitination domain. Whereas each mutation similarly disrupted nuclear morphology, nondisjunction was less frequent in Df(114-118) mutants, suggesting that nondisjunction is not a direct consequence of nuclear perturbation. Anaphase I bridges were observed in both C105A and Df(114-118) mutants, but at a much lower frequency in Df(114-118) mutants, suggesting that ubiquitination of targets important for chromosome segregation still occurs in Df(114-118) males. Bridge formation was independent of the sister chromatid cohesin Sunn, indicating that bridges did not result from sister chromatid attachments. In yeast two hybrid assays, dTopors interacted with six of the 28 E2 ubiquitin-conjugating enzymes in Drosophila: CG10862, Ubc2, CG7220, Taf1, CG8188, and Effete. Interactions were abolished by C105A, but only diminished by Df(114-118). We suggest that interaction of Df(114-118) with one or more E2s is nearly sufficient for ubiquitination of target(s) required for chromosome segregation but not nuclear structure. Comparative proteomic analysis by mass spectroscopy identified 924 proteins differentially ubiquitinated in C105A vs wildtype testis, including histones and Lamin Dm0. The largest ontology group of proteins identified, however, was components of ubiquitin-mediated proteolysis, suggesting that dTopors may also play a role in coordinating pathways that regulate protein turnover.
