| SUMO (small ubiquitin-like modifier protein) is a small protein that influences the biochemical and consequently physiological behavior of a large number of proteins through covalent attachment to these proteins. To investigate the roles of SUMO in Drosophila embryo development, I generated and examined maternal germline clones of sumo hypomorphic alleles to reduce the level of maternally contributed SUMO in the embryo. Phenotypic analysis revealed possible involvement of SUMO in EGFR/Ras signaling, cell cycle control, and pattern formation.;To facilitate a mechanistic understanding of the diverse roles of SUMO early development, I affinity purified and identified SUMO conjugation targets from transgenic flies expressing dual-tagged SUMO, leading to the identification of 142 potential sumoylation targets. Enriched functional groups included groups of proteins regulating embryonic patterning, translation and localization of maternal mRNAs, cell cycle regulation, and Ras signaling. Using a bacterial sumoylation system, I verified a number of the proteins identified in the proteomic screen as sumoylation substrates.;The roles of SUMO in Ras signaling and cell cycle regulation, as revealed by the phenotypic analysis of sumo mutants, were confirmed in additional studies. SUMO is required for Ras signaling upstream of or in parallel with Ras1 activation. The requirement of SUMO in cell cycle progression was demonstrated by RNA interference-mediated knockdown of SUMO in cultured Drosophila cells, or in larval tissues. During mitosis, SUMO was found to localize to the centromere-kinetochore region and the spindle midbody, further suggesting essential functions for sumoylation throughout mitosis. Colocalization between SUMO and Ubc9, as well as partial colocalization between SUMO and Polo, one of the proteins identified in the proteomic screen, were observed at the kinetochore, suggesting that sumoylation of multiple kinetochore proteins ensures proper mitotic spindle attachment and/or spindle checkpoint control. |
