Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners.
dc.contributor.author | Yu, Seok-Ho | |
dc.contributor.author | Boyce, Michael | |
dc.contributor.author | Wands, Amberlyn M | |
dc.contributor.author | Bond, Michelle R | |
dc.contributor.author | Bertozzi, Carolyn R | |
dc.contributor.author | Kohler, Jennifer J | |
dc.date.accessioned | 2020-01-01T17:03:41Z | |
dc.date.available | 2020-01-01T17:03:41Z | |
dc.date.issued | 2012-03-12 | |
dc.date.updated | 2020-01-01T17:03:40Z | |
dc.description.abstract | O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation. | |
dc.identifier | 1114356109 | |
dc.identifier.issn | 0027-8424 | |
dc.identifier.issn | 1091-6490 | |
dc.identifier.uri | ||
dc.language | eng | |
dc.publisher | Proceedings of the National Academy of Sciences | |
dc.relation.ispartof | Proceedings of the National Academy of Sciences of the United States of America | |
dc.relation.isversionof | 10.1073/pnas.1114356109 | |
dc.subject | Hela Cells | |
dc.subject | Cell Nucleus | |
dc.subject | Humans | |
dc.subject | Diazomethane | |
dc.subject | Acetylglucosamine | |
dc.subject | Peptides | |
dc.subject | Nuclear Pore Complex Proteins | |
dc.subject | Uridine Diphosphate | |
dc.subject | Cross-Linking Reagents | |
dc.subject | Staining and Labeling | |
dc.subject | Protein Processing, Post-Translational | |
dc.subject | Mutagenesis | |
dc.subject | Repetitive Sequences, Amino Acid | |
dc.subject | Protein Binding | |
dc.subject | Active Transport, Cell Nucleus | |
dc.subject | Light | |
dc.subject | Models, Biological | |
dc.title | Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners. | |
dc.type | Journal article | |
duke.contributor.orcid | Boyce, Michael|0000-0002-2729-4876 | |
pubs.begin-page | 4834 | |
pubs.end-page | 4839 | |
pubs.issue | 13 | |
pubs.organisational-group | School of Medicine | |
pubs.organisational-group | Duke | |
pubs.organisational-group | Duke Cancer Institute | |
pubs.organisational-group | Institutes and Centers | |
pubs.organisational-group | Biochemistry | |
pubs.organisational-group | Basic Science Departments | |
pubs.publication-status | Published | |
pubs.volume | 109 |
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