Identification and characterization of elements of a Drosophilaubiquitin-like-protein conjugation pathway that may be involved in septin modification. H.-P. Shih , K.G. Hales , M. Peifer , J.R. Pringle. Department of Biology and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC.

   In a two-hybrid screen designed to identify septin-interacting proteins, Drosophila homologues of both yeast UBA2 (Dmuba2) and UBC9 (Dmubc9) were identified. In Saccharomyces cerevisiae, the heterodimer of Uba2p and Aos1p serves as the activating enzyme (E1-type enzyme) and Ubc9p is the conjugating enzyme (E2-type enzyme) for the ubiquitin-like protein Smt3p. Smt3p is transferred to target proteins through this protein-conjugation pathway, and recent data indicate that yeast septins are Smt3p modified, although the function(s) of this modification are not yet clear. We have cloned Dmuba2, Dmubc9, and Drosophila homologues of AOS1 (Dmaos1) and SMT3 (Dmsmt3). DmUba2 and DmAos1 interact with each other in the two-hybrid system. We have also shown that DmUba2 and DmUbc9 interact with DmSmt3, and these interactions are via the expected thiolester bonds. Furthermore, DmUba2 and DmUbc9 can be co-immunoprecipitated. Therefore, we conclude that DmUba2/DmAos1 and DmUbc9 act as E1-type and E2-type enzymes, respectively for DmSmt3, implying that this ubiquitin-like-protein conjugation pathway is well conserved in Drosophila. Whether Drosophila septins are modified by DmSmt3p is under investigation. If so, the function of such modification will be studied further.