In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy.
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.
Matic
Ivan
I
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
van Hagen
Martijn
M
Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
Schimmel
Joost
J
Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
Macek
Boris
B
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Ogg
Stephen C
SC
Centre for Molecular Medicine, 61 Biopolis Drive (Proteos), Singapore 138673, Singapore.
Tatham
Michael H
MH
Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
Hay
Ronald T
RT
Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
Lamond
Angus I
AI
Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
Mann
Matthias
M
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Vertegaal
Alfred C O
ACO
Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
D000595:Amino Acid Sequence; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D025842:SUMO-1 Protein / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D002457:Cell Extracts; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010455:Peptides; D011108:Polymers; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins