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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M700173-MCP200v1 7/1/132    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Glossary Google Scholar Articles by Matic, I. Articles by Vertegaal, A. C. O. PubMed PubMed Citation Articles by Matic, I. Articles by Vertegaal, A. C. O.

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Research

In Vivo Identification of Human Small Ubiquitin-like Modifier Polymerization Sites by High Accuracy Mass Spectrometry and an in Vitro to in Vivo Strategy^*,S

Ivan Matic^,, Martijn van Hagen^,¶, Joost Schimmel^¶, Boris Macek, Stephen C. Ogg^||, Michael H. Tatham^**, Ronald T. Hay^**, Angus I. Lamond^**,, Matthias Mann^, and Alfred C. O. Vertegaal^¶,¶¶

From the Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany, ^¶ Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands, ^|| Centre for Molecular Medicine, 61 Biopolis Drive (Proteos), Singapore 138673, Singapore, and ^** Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.

^¶¶ To whom correspondence may be addressed. Tel.: 31-71-526-9621; Fax: 31-7-526-8270; E-mail: vertegaal{at}lumc.nl