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Amino Acid Sequence
Cell Extracts
Cell Nucleus/metabolism
HeLa Cells
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/chemistry/metabolism
Mass Spectrometry
Molecular Sequence Data
Peptides/chemistry
Polymers/*chemistry
SUMO-1 Protein/chemistry/isolation & purification/metabolism
Small Ubiquitin-Related Modifier Proteins/*chemistry/isolation &
purification/metabolism
Ubiquitins/chemistry/isolation & purification/metabolism
Abstract:
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.
