Abstract
SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.
Highlights
Post-translational protein modification adds layers of complexity to macromolecular function
To determine the early embryonic SUMO-ome, we adopted a scheme that involved a twostep affinity purification strategy using SUMO tagged at its Nterminus with both (His)6 and FLAG tags, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protein identification of trypsin-digested proteins (Figure 1A)
Since the SUMO conjugates were in low abundance even when very large amount of starting materials were used, we promoted SUMO conjugation with heat treatment at 37uC
Summary
Post-translational protein modification adds layers of complexity to macromolecular function. One way of modifying proteins is by joining the ubiquitin family proteins to lysine residues, generating branched proteins [1]. One such ubiquitin-like protein, SUMO (small ubiquitin-related modifier), displays remarkable versatility in modulating target protein function. Many proteins are targeted for covalent modification by SUMO, which modulates many cellular processes [2,3,4]. In S. cerevisiae, mutations in genes encoding SUMO pathway enzymes are lethal [5,6,7], while mutations in the corresponding genes in S. pombe severely impair growth [8,9,10]. Deletion of genes encoding enzymes required for SUMO conjugation in C. elegans leads to embryonic lethality [11], while reduction of the SUMO conjugating enzyme levels in Drosophila, zebrafish, and mouse results in developmental defects [12,13,14]
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