Covalent modification of the transcriptional repressor tramtrack by the ubiquitin-related protein Smt3 in Drosophila flies

The ubiquitin-related SUMO-1 modifier can be covalently attached to a varie ty of proteins, To date, four substrates have been characterized in mammali an cells: RanGAP1, I kappa B alpha, and the two nuclear body-associated PML and Sp100 proteins, SUMO-1 modification has been shown to be involved in p rotein localization and/or stabilization and to require the activity of spe cialized E1-activating and E2 Ubc9-conjugating enzymes. SUMO-1 homologues h ave been identified in various species and belong to the so-called Smt3 fam ily of proteins. Here we have characterized the Drosophila homologues of ma mmalian SUMO-1 and Ubc9 (termed dSmt3 and dUbc9, respectively). We show tha t dUbc9 is the conjugating enzyme for dSmt3 and, that dSmtB can covalently modify a number of proteins in Drosophila cells in addition to the human PM L substrate. The dSmt3 transcript and protein are maternally deposited in e mbryos, where the protein accumulates predominantly in nuclei, Similar to i ts human counterpart, dSmt3 protein is observed in a punctate nuclear patte rn. Mie demonstrate that Tramtrack 69 (Ttk69), a repressor of neuronal diff erentiation, is a bona fide in vivo substrate for dSmt3 conjugation. Finall y, we show that both the modified and unmodified forms of Ttk69 can bind to a Ttk69 binding site in vitro. Moreover, dSmt3 and Ttk69 proteins colocali ze on polytene chromosomes, indicating that the dSmt3-conjugated Ttk69 spec ies can bind at sites of Ttk69 action in vivo. Altogether, these data indic ate a high conservation of the Smt3 conjugation pathway and further suggest that this mechanism may play a role in the transcriptional regulation of c ell differentiation in Drosophila flies.