Identification of septin-interacting proteins and characterization of the Smt3/SUMO-conjugation system in Drosophila.
The septins are a family of proteins involved in cytokinesis and other aspects of cell-cortex organization. In a two-hybrid screen designed to identify septin-interacting proteins in Drosophila, we isolated several genes, including homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9. Yeast Uba2p and Ubc9p are involved in the activation and conjugation, respectively, of the ubiquitin-like protein Smt3p/SUMO, which becomes conjugated to a variety of proteins through this pathway. Uba2p functions together with a second protein, Aos1p. We also cloned and characterized the Drosophila homologues of AOS1 (Dmaos1) and SMT3 (Dmsmt3). Our biochemical data suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating enzymes for DmSmt3, implying that this protein-conjugation pathway is well conserved in Drosophila. Immunofluorescence studies showed that DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial blastoderm stage. In older embryos, DmUba2 and DmSmt3 are both concentrated in the nuclei during interphase but dispersed throughout the cells during mitosis, with DmSmt3 also enriched on the chromosomes during mitosis. These data suggest that DmSmt3 could modify target proteins both inside and outside the nuclei. We did not observe any concentration of DmUba2 at sites where the septins are concentrated, and we could not detect DmSmt3 modification of the three Drosophila septins tested. However, we did observe DmSmt3 localization to the midbody during cytokinesis both in tissue-culture cells and in embryonic mitotic domains, suggesting that DmSmt3 modification of septins and/or other midzone proteins occurs during cytokinesis in Drosophila.
Shih
Hsin-Pei
HP
Department of Biology, University of North Carolina, Chapel Hill, NC 27599 USA.
Hales
Karen G
KG
Pringle
John R
JR
Peifer
Mark
M
D000818:Animals; D002478:Cells, Cultured; D002999:Clone Cells; D004330:Drosophila / Q000166:cytology / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D007399:Interphase; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008938:Mitosis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D020539:Sequence Analysis, Protein; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D029721:Drosophila Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C099362:SMT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C490960:smt3 protein, Drosophila; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D019204:GTP-Binding Proteins; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes; C429037:uba2 protein, Drosophila; C404950:Aos1 protein, Drosophila; D000098843:Ubiquitin-Conjugating Enzyme UBC9