Abstract
The SUMO family in vertebrates includes at least three distinct proteins (SUMO-1, -2, and -3) that are added as post-translational modifications to target proteins. A considerable number of SUMO-1 target proteins have been identified, but little is known about SUMO-2. A stable HeLa cell line expressing His6-tagged SUMO-2 was established and used to label and purify novel endogenous SUMO-2 target proteins. Tagged forms of SUMO-2 were functional and localized predominantly in the nucleus. His6-tagged SUMO-2 conjugates were affinity-purified from nuclear fractions and identified by mass spectrometry. Eight novel potential SUMO-2 target proteins were identified by at least two peptides. Three of these proteins, SART1, heterogeneous nuclear ribonucleoprotein (RNP) M, and the U5 small nuclear RNP 200-kDa helicase, play a role in RNA metabolism. SART1 and heterogeneous nuclear RNP M were both shown to be genuine SUMO targets, confirming the validity of the approach.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antigens, Neoplasm / metabolism
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Bacterial Proteins / genetics
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Cell Nucleus / chemistry
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Chromatography, High Pressure Liquid
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Gene Expression
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HeLa Cells
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Heterogeneous-Nuclear Ribonucleoprotein Group M / metabolism
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Humans
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Luminescent Proteins / genetics
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Mass Spectrometry
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Microscopy, Fluorescence
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Protein Processing, Post-Translational
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Proteomics*
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Receptors, N-Acetylglucosamine / metabolism
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Recombinant Fusion Proteins
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Ribonucleoprotein, U5 Small Nuclear / metabolism
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Ribonucleoproteins, Small Nuclear / metabolism
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Small Ubiquitin-Related Modifier Proteins / analysis
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Small Ubiquitin-Related Modifier Proteins / genetics
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Small Ubiquitin-Related Modifier Proteins / metabolism*
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Transfection
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Trypsin / metabolism
Substances
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Antigens, Neoplasm
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Bacterial Proteins
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HNRNPM protein, human
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Heterogeneous-Nuclear Ribonucleoprotein Group M
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Luminescent Proteins
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Receptors, N-Acetylglucosamine
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Recombinant Fusion Proteins
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Ribonucleoprotein, U5 Small Nuclear
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Ribonucleoproteins, Small Nuclear
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SART1 protein, human
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Small Ubiquitin-Related Modifier Proteins
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yellow fluorescent protein, Bacteria
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Trypsin