| The septins are a family of proteins involved in cytokinesis and other aspects of cell surface organization. I set out to identify septin-associated proteins, in order to learn more about septin functions. I utilized the two-hybrid system and identified Drosophila homologues of both yeast UBA2 (Dmuba2) and UBC9 ( Dmubc9) as septin interactors. 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 proteinconjugation pathway, and recent data indicate that yeast septins are Smt3p modified, although the function(s) of this modification are not yet clear. We 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 that 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, suggesting that this ubiquitin-like-protein conjugation pathway is well conserved in Drosophila. We also analyzed the localization of DmUba2 and DmSmt3 during embryonic and postembryonic development. The localization of DmSmt3 at the midbody during cytokinesis raised the possibility that septins or midzone proteins may be modified by DmSmt3.; In Drosophila there are five septins: Pnut, Sep1, Sep2, Sep4, and Sep5. Previous studies showed that septins function as a single complex in certain cellular events. However, other evidence does not favor this concept. For example, the localization of Sep2 does not depend on Pnut in cellularization. To extend the previous work on Sep2, I took advantage of a sep2 mutant to test the specificity of Sep2 antibody. I also assessed the localization of GFP (Green Fluorescent Protein)-tagged Sep2 in living embryos during cellularization and embryonic cytokinesis. Another unsolved issue concerning septins is the relationship between the GTPase motif and septin functions. I ectopically expressed a putative GTP-binding defective mutant pnut (S156N) and examined the consequence of this expression. |
