Roles for Drosophila Smt3 in the response to stress. M.T. Smith , V. Bhaskar , A.J. Courey. Chemistry & Biochemistry, University of California at Los Angeles, Los Angeles, CA.

   Smt3 (also called SUMO) is a ubiquitin-like protein that can be covalently conjugated to target proteins in a manner similar to ubiquitin. We have previously shown that the rel family transcription factor, Dorsal, is a substrate for Smt3 conjugation. This conjugation event appears to enhance Dorsal activity, at least in part, by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. The Smt3 conjugation machinery also stimulates Dorsal activity under conditions in which Dorsal is constitutively nuclear, suggesting that conjugation may directly influence the ability of Dorsal to activate transcription. Smt3 conjugation occurs at a single site in Dorsal (lysine 382) and mutagenesis of this acceptor lysine results in a form of Dorsal that displays enhanced levels of synergistic transcriptional activation. In addition to controlling dorsoventral pattern formation during embryogenesis, Dorsal has been shown to play a role in mediating the innate immune response. We found that components of the Smt3 conjugation pathway are required for lipopolysaccharide-induced expression of anti-microbial peptides in cultured cells and larvae.
   Our analysis of the Smt3 conjugation pathway has shown it involves an E1-like activating enzyme (Sae1/Sae2) and an E2-like conjugating enzyme (Ubc9), which together are sufficient for conjugating Smt3 to Dorsal, in vitro. We have also characterized an Smt3-specific cysteine protease (Ulp1) that is responsible for deconjugating Smt3 from target proteins and for processing Smt3 into its active form. Ulp1 is predominately localized at the nuclear periphery and it appears to play a role in transporting Smt3 into the nucleus. We are currently evaluating Smt3 conjugation as a general stress response mechanism. Additionally, we are characterizing a novel Smt3 conjugation target and we are assessing a potential E3-like Smt3 ligase candidate.