Small ubiquitin-like modifier (SUMO) is conjugated to its substrates via an enzymatic cascade consisting of three enzymes, E1, E2, and E3. The active site of the E2 enzyme, Ubc9, recognizes the substrate through binding to a consensus tetrapeptide ⌿KXE. However, recent proteomics studies suggested that a considerable part of sumoylation occurs on non-consensus sites. Current unbiased sumoylation site identification techniques typically require high stoichiometry in vitro sumoylation, mass spectrometry, and complex data analysis. To facilitate in vivo analysis, we have designed a mass spectrometric method based on an engineered human SUMO-1 construct that creates a signature tag on SUMO substrates. This construct enables affinity purification by covalent binding to cysteine residues in LysC/trypsin-cleaved peptides and site identification by diglycyl lysine tagging of sumoylation sites. As a proof of concept, site-specific and substrate-unbiased in vivo sumoylation analysis of HeLa cells was performed. We identified 14 sumoylation sites, including well known sites, such as Lys 524 of RanGAP1, and novel non-consensus sites. Only 3 of the 14 sites matched consensus sites, supporting the emerging view that nonconsensus sumoylation is a common event in live cells. Six of the non-consensus sites had a nearby SUMO interaction motif (SIM), which emphasizes the role of SIM in non-consensus sumoylation. Nevertheless, the lack of nearby SIM residues among the remaining non-consensus sites indicates that there are also other specificity determinants of non-consensus sumoylation. The method we have developed proved to be a useful tool for sumoylation studies and will facilitate identification of novel SUMO substrates containing both consensus and non-consensus sites.Sumoylation is a post-translational modification that consists of covalent conjugation of the small ubiquitin-like modifier (SUMO) 5 to substrate proteins and results in altered activity of the substrate. Sumoylation influences a plethora of cellular processes, including transcriptional regulation of gene expression and genome integrity (1). SUMO conjugation involves an enzymatic cascade employing three enzymes: activating E1, conjugating E2, and ligating E3 (2). Because the active site of the single E2, Ubc9, recognizes the substrate through binding to a consensus tetrapeptide ⌿KXE (⌿, a hydrophobic amino acid; K, the target lysine, X, any amino acid, and E, glutamic acid), SUMO acceptor sites are to date predominantly identified through mutagenesis of target lysine residues on consensus tetrapeptides (3). Recent proteomics studies by us and others have shown that a considerable proportion of sumoylated proteins do not contain the consensus sites (4 -6) and are unreachable by the conventional mutagenesis approach. Furthermore, a model for non-consensus SUMO targeting has been proposed, where SUMO-Ubc9 thioester is recruited by a SUMO interaction motif (SIM) located on the substrate (7). However, the mechanisms for targeting non-consensus substrates remain larg...
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